Lipid formulations for delivery of messenger RNA

ABSTRACT

The present invention provides, among other things, methods of delivering mRNA in vivo, including administering to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in vivo, wherein the liposome comprises a cationic lipid of formula I-c: 
                         
or a pharmaceutically acceptable salt thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/026,577, filed Jul. 3, 2018; which is continuation application of U.S. application Ser. No. 15/451,312, filed Mar. 6, 2017; which is a divisional application of U.S. application Ser. No. 14/521,161, filed Oct. 22, 2014; which claims priority to U.S. Provisional Application Ser. No. 61/894,299, filed Oct. 22, 2013 and U.S. Provisional Application Ser. No. 61/953,516, filed Mar. 14, 2014, the disclosures of which are hereby incorporated by reference.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing (submitted electronically as .txt file named “MRT-1121US4 MIT17255 SL.txt” on Nov. 14, 2019). The .txt file was generated on Nov. 14, 2019 and is 32,585 bytes in size. The entire contents of the Sequence Listing are herein incorporated by reference.

BACKGROUND

Delivery of nucleic acids has been explored extensively as a potential therapeutic option for certain disease states. In particular, RNA interference (RNAi) has been the subject of significant research and clinical development. While RNAi, such as short interfering RNA (siRNA), may have therapeutic potential, it is of little use in treating diseases involving deficiency of one or more proteins. messenger RNA (mRNA) therapy has become an increasingly important option for treatment of various diseases, in particular, for those associated with deficiency of one or more proteins.

SUMMARY OF THE INVENTION

The present invention provides improved methods and compositions for highly efficient delivery and expression of mRNA and encoded protein in vivo. The invention is based, in part, on the surprising discovery that liposomes based on a particular class of cationic lipids, such as, those having a structure of formula I-c described herein, are unexpectedly effective in delivering mRNA and producing encoded protein in vivo, more effective even as compared to those cationic lipids that were considered to be among the best in delivering mRNA in the prior art. Indeed, prior to the present invention, cationic lipids have been extensively explored as an important component of liposomes typically used to encapsulate nucleic acids including mRNA for in vivo delivery. Due to the uniquely fragile and long structure of mRNA and the complicated in vivo translation process, cationic lipids used in the liposomes typically play two roles. First, cationic lipids promote interaction with negatively charged mRNA during encapsulation, circulation and endocytosis, thereby capturing and protecting the mRNA. Then, once inside cytosol, cationic lipids need to be able to release the mRNA so that the mRNA can be translated to produce encoded protein. Some cationic lipids, in particular, those known as titratable cationic lipids are particularly effective in delivering mRNA. One example of such cationic lipids known to be capable of efficient delivery of mRNA is C12-200. Surprisingly, the present inventors found that cationic lipids described herein can be even more effective in delivering various mRNA in vivo, than those best known in the prior art including C12-200. For example, as shown in the Examples below, liposome particles incorporating a cationic lipid described herein (e.g., cKK-E12) resulted in close to 50% higher protein expression of human Factor IX protein detected in the plasma of administered mice, as compared to C12-200-based liposome particles. Furthermore, the plasma residence time of different proteins expressed from mRNA delivered by cKK-E12 based liposomes is sustained up to 7 days or longer post a single administration. Thus, the present inventors have demonstrated that this class of cationic lipids having a structure of formula I-c described herein (e.g., cKK-E12) can be uniquely useful in delivering mRNA for highly efficient and sustained production of protein (e.g., therapeutic protein) in vivo. The present invention therefore permits an improved mRNA therapy that can significantly reduce required amount of mRNA and associated lipids, administration frequency, and possible side effects, providing more potent, safer, and patient friendly mRNA therapy for various diseases.

In one aspect, the present invention provides methods of delivering messenger RNA (mRNA) in vivo, including administering to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in vivo, wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   p is an integer of between 1 and 9, inclusive;     -   each instance of R² is independently hydrogen or optionally         substituted C₁₋₆ alkyl;     -   each instance of R⁶ and R⁷ is independently a group of the         formula (i), (ii), or (iii);     -   Formulae (i), (ii), and (iii) are:

wherein:

-   -   each instance of R′ is independently hydrogen or optionally         substituted alkyl;     -   X is O, S, or NR^(X), wherein R^(X) is hydrogen, optionally         substituted alkyl, optionally substituted alkenyl, optionally         substituted alkynyl, optionally substituted carbocyclyl,         optionally substituted heterocyclyl, optionally substituted         aryl, optionally substituted heteroaryl, or a nitrogen         protecting group;     -   Y is O, S, or NR^(Y), wherein R^(Y) is hydrogen, optionally         substituted alkyl, optionally substituted alkenyl, optionally         substituted alkynyl, optionally substituted carbocyclyl,         optionally substituted heterocyclyl, optionally substituted         aryl, optionally substituted heteroaryl, or a nitrogen         protecting group;     -   R^(P) is hydrogen, optionally substituted alkyl, optionally         substituted alkenyl, optionally substituted alkynyl, optionally         substituted carbocyclyl, optionally substituted heterocyclyl,         optionally substituted aryl, optionally substituted heteroaryl,         an oxygen protecting group when attached to an oxygen atom, a         sulfur protecting group when attached to a sulfur atom, or a         nitrogen protecting group when attached to a nitrogen atom; and     -   R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally         substituted C₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl,         optionally substituted heteroC₁₋₅₀ alkyl, optionally substituted         heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl,         or a polymer.

In another aspect, the present invention provides methods of treating a disease or disorder including administering to subject in need of treatment a composition comprising an mRNA encoding a therapeutic protein encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in one or more tissues affected by the disease or disorder, wherein the liposome comprises a cationic lipid having a structure of formula I-c.

In another aspect, the present invention provides compositions for delivery of messenger RNA (mRNA) comprising an mRNA encoding a protein encapsulated within a liposome, wherein the liposome comprises a cationic lipid having a structure of formula I-c.

In some embodiments, a suitable cationic lipid is cKK-E12:

In some embodiments, a suitable liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids. In some embodiments, the one or more non-cationic lipids are selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof.

In some embodiments, a suitable liposome further comprises one or more cholesterol-based lipids. In some embodiments, the one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol and DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine.

In some embodiments, a suitable liposome further comprises one or more PEG-modified lipids. In some embodiments, the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C₆-C₂₀ length. In some embodiments, a PEG-modified lipid is a derivatized ceramide such as N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000]. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or Dimyristoylglycerol (DMG)-PEG-2K.

In some embodiments, a suitable liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.

In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio. In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%, about 35%, about 40%, about 45%, or about 50% of the liposome by molar ratio.

In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by molar ratio. In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:25:5 by molar ratio. In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by molar ratio.

In some embodiments, a suitable liposome has a size of or less than about 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 150 nm, 125 nm, 110 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50 nm.

In some embodiments, a composition according to the invention is administered intravenously. In some embodiments, a composition according to the invention is administered via pulmonary delivery. In some embodiments, the pulmonary delivery is by aerosolization, inhalation, nebulization or instillation. In some embodiments, a composition according to the invention is administered intrathecally. In some embodiments, the composition is formulated as respirable particles, nebulizable lipid, or inhalable dry powder.

In some embodiments, the expression of the protein encoded by the mRNA is detectable in liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.

In some embodiments, the expression of the protein encoded by the mRNA is detectable 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or 72 hours after the administration. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and/or 7 days after the administration. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks after the administration. In some embodiments, the expression of the protein encoded by the mRNA is detectable after a month after the administration.

In some embodiments, the protein encoded by the mRNA is a cytosolic protein. In some embodiments, the protein encoded by the mRNA is a secreted protein. In some embodiments, the protein encoded by the mRNA is an enzyme. In some embodiments, the mRNA has a length of or greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb. In some embodiments, the protein encoded by the mRNA is Argininosuccinate Synthetase (ASS1), Factor IX, survival of motor neuron 1, or phenylalanine hydroxylase.

In some embodiments, the mRNA is administered at a dose ranging from about 0.1-5.0 mg/kg body weight, for example about 0.1-4.5, 0.1-4.0, 0.1-3.5, 0.1-3.0, 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3, 0.3-5.0, 0.3-4.5, 0.3-4.0, 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, 0.3-0.5, 0.5-5.0, 0.5-4.5, 0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, or 0.5-1.0 mg/kg body weight. In some embodiments, the mRNA is administered at a dose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg body weight.

In some embodiments, the mRNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides comprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine. In some embodiments, the mRNA is unmodified.

Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWING

The drawings are for illustration purposes only not for limitation.

FIG. 1 shows an exemplary graph of the levels of human factor IX (FIX) detected in the serum of treated mice 24 hours after administration of C12-200 or cKK-E12 liposomes containing FIX mRNA.

FIG. 2 shows an exemplary graph of FIX detected in the plasma of mice treated with 0.1, 0.3, 0.6, 1.0, or 3.0 mg/kg of one of two ratios of FIX mRNA containing cKK-E12 liposomes either 6 or 24 hours after administration.

FIG. 3 shows an exemplary graph of the level of ASS1 protein detected in the livers of mice treated with 0.1, 0.3, 0.6, 1.0, or 3.0 mg/kg of ASS1 mRNA-containing cKK-E12 liposomes 24 hours after administration.

FIG. 4 shows exemplary western blot analyses of ASS1 protein levels in the liver 24 hours post administration of 0.1, 0.3, 0.6, 1.0, or 3.0 mg/kg of cKK-E12 liposomes containing ASS1 mRNA.

FIG. 5 shows an exemplary graph of ASS1 protein levels in the liver of mice 0.5, 3, 6, 12, 24, 48, 72 hours after a single IV injection of ASS1 mRNA containing cKK-E12 liposomes (1 mg/kg). Also shown is the level of ASS1 protein 7 days after administration.

FIG. 6 shows exemplary western blot analyses of ASS1 protein levels in the liver 0.5, 3, 6, 12, 24, 48, 72 hours after a single IV injection of 1 mg/kg ASS1 mRNA containing cKK-E12 liposomes. Also shown is the level of ASS1 protein 7 days after administration.

FIG. 7—shows detection of human ASS1 messenger RNA via in situ hybridization in the livers of treated mice. Exogenous mRNA is observable for at least 72 hr post-administration after a single dose (1.0 mg/kg) of ASS1 mRNA-loaded MD1-based lipid nanoparticles. Human ASS1 mRNA is detectable in sinusoidal cells as well as hepatocytes.

FIG. 8—shows exemplary immunohistochemical staining of ASS1 protein levels in mouse liver 24 hours after administration of 1 mg/kg ASS1 mRNA containing cKK-E12 lipid nanoparticles. Human ASS1 protein is detectable in sinusoidal cells as well as hepatocytes.

FIG. 9 shows low magnification (4×) immunohistochemical staining of ASS1 protein levels in mouse liver 24 hours after administration of 1 mg/kg ASS1 mRNA containing cKK-E12 liposomes. A comparison to untreated mouse liver (left) demonstrates the widespread distribution of human ASS1 protein throughout the liver.

FIG. 10 shows exemplary results illustrating that cKK-E12 lipid nanoparticles efficiently delivered FL mRNA via nebulization. Mice were exposed to milligram of encapsulated FL mRNA and analysis was performed 24 hours post-exposure.

FIG. 11 illustrates detection via western blot of human SMN-1 protein derived from exogenous hSMN-1 mRNA that was transfected into BHK-21 cells. Various antibodies specific to human SMN were employed: (A) anti-SMN 4F11 antibody at 1:1,000 dilution; (B) Pierce PA5-27309 a-SMN antibody at 1:10,000 dilution; and (C) LSBio C138149 a-SMN antibody at 1:10,000 dilution.

FIG. 12A-C illustrates multiplex nucleic acid in situ detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A) Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post intrathecal delivery.

FIG. 13 illustrates positive detection of human SMN-1 protein produced in the spinal cord of a rat 24 hours post-intrathecal administration of human SMN-1 mRNA-loaded lipid nanoparticles. Anti-human SMN 4F11 antibody was employed at 1:2500 dilution. Panel A represents treated rat spinal cord tissue and panel B represents untreated rat spinal cord tissue.

FIG. 14 In vivo transfection of CFTR knockout mice with C-terminal His₁₀ tagged (SEQ ID NO: 11) codon-optimized human CFTR mRNA encapsulated within either a lipid (cKK-E12) or polymeric (PEI) nanoparticle formulation. Following nebulized delivery of each respective mRNA formulation, Right and Left lung tissue lysate was collected and analyzed for CFTR expression by Western blot using anti-His antibody 1187. Control CFTR knockout lung tissue and CFTR-His₁₀ HEK293 lysate (“His₁₀” disclosed as SEQ ID NO: 11) was used as a negative and positive controls respectively.

FIG. 15 illustrates positive detection of active firefly luciferase (FFL) protein in a treated pig lung via luminescence upon exposure to FFL/CO-CFTR—C-His₁₀ mRNA (“His₁₀” disclosed as SEQ ID NO: 11) encapsulated cKK-E12 lipid nanoparticles. Pigs were treated with 1 mg FFL+9 mg CO-CFTR—C-His₁₀ mRNA (“His₁₀” disclosed as SEQ ID NO: 11) encapsulated lipid nanoparticles via nebulization using a Pari jet nebulizer and sacrificed 24 hours post-treatment. FFL luminescence was visualized using an IVIS bioluminometer.

DEFINITIONS

In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

Amino acid: As used herein, term “amino acid,” in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H_(E)N—C(H)(R)—COHO. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid. “Standard amino acid” refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, “synthetic amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxyl- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (aslo referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).

Expression: As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody). In this application, the terms “expression” and “production,” and grammatical equivalent, are used inter-changeably.

Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

Half-life: As used herein, the term “half-life” is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.

Improve, increase, or reduce: As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

In Vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).

Local distribution or delivery: As used herein, the terms “local distribution,” “local delivery,” or grammatical equivalent, refer to tissue specific delivery or distribution. Typically, local distribution or delivery requires a protein (e.g., enzyme) encoded by mRNAs be translated and expressed intracellularly or with limited secretion that avoids entering the patient's circulation system.

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine (“5mC”), pseudouridine (“pU”), and/or 2-thio-uridine (“2sU”). See, e.g., U.S. Pat. No. 8,278,036 or WO2011012316 for a discussion of such residues and their incorporation into mRNA. The mRNA may be RNA, which is defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings for the use of RNA are disclosed US Patent Publication US20120195936 and internation publication WO2011012316, both of which are hereby incorporated by reference in their entirety. The presence of nonstandard nucleotide residues may render an mRNA more stable and/or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In further embodiments, the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications. Certain embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2′-O-alkyl modification, a locked nucleic acid (LNA)). In some embodiments, the RNAs may be complexed or hybridized with additional polynucleotides and/or peptide polynucleotides (PNA). In embodiments where the sugar modification is a 2′-O-alkyl modification, such modification may include, but are not limited to a 2′-deoxy-2′-fluoro modification, a 2′-O-methyl modification, a 2′-O-methoxyethyl modification and a 2′-deoxy modification. In certain embodiments, any of these modifications may be present in 0-100% of the nucleotides—for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.

Patient: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre and post natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Systemic distribution or delivery: As used herein, the terms “systemic distribution,” “systemic delivery,” or grammatical equivalent, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream. Compared to the definition of “local distribution or delivery.”

Subject: As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Target tissues: As used herein, the term “target tissues” refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

DETAILED DESCRIPTION

The present invention provides, among other things, methods and compositions for delivering mRNA in vivo using improved liposomes incorporating cationic lipids described herein.

Liposomes for mRNA Delivery

As used herein, the term “liposome” refers to any lamellar, multilamellar, or solid lipid nanoparticle vesicle. Typically, a liposome as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s). Thus, the term “liposome” as used herein encompasses both lipid and polymer based nanoparticles. In particular, a liposome according to the present invention incorporates a cationic lipid described herein. As a non-limiting example, a cationic lipid suitable for the present invention is cKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), as described in more detail below. A suitable liposome may also contain second or additional cationic lipids, helper lipids (e.g., non-cationic lipids and/or cholesterol-based lipids), PEG-modified lipids, and/or polymers.

In some embodiments, cationic lipid(s) (e.g., cKK-E12) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio. In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%, about 35%, about 40%, about 45%, or about 50% of the liposome by molar ratio.

Cationic Lipids

In some embodiments, provided liposomes or compositions provided comprise a cationic lipid according to formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   p is an integer of between 1 and 9, inclusive;     -   each instance of Q is independently O, S, or NR^(Q);     -   R^(Q) is hydrogen, optionally substituted alkyl, optionally         substituted alkenyl, optionally substituted alkynyl, optionally         substituted carbocyclyl, optionally substituted heterocyclyl,         optionally substituted aryl, optionally substituted heteroaryl,         a nitrogen protecting group, or a group of the formula (i), (ii)         or (iii);     -   each instance of R¹ is independently hydrogen, optionally         substituted alkyl, optionally substituted alkenyl, optionally         substituted alkynyl, optionally substituted carbocyclyl,         optionally substituted heterocyclyl, optionally substituted         aryl, optionally substituted heteroaryl, halogen, —OR^(A1),         —N(R^(A1))₂, —SR^(A1), or a group of formula (iv):

-   -   L is an optionally substituted,alkylene, optionally substituted         alkenylene, optionally substituted alkynylene, optionally         substituted heteroalkylene, optionally substituted         heteroalkenylene, optionally substituted heteralkynylene,         optionally substituted carbocyclylene, optionally substituted         heterocyclylene, optionally substituted arylene, or optionally         substituted heteroarylene, or combination thereof, and     -   each of R⁶ and R⁷ is independently hydrogen, optionally         substituted alkyl, optionally substituted alkenyl, optionally         substituted alkynyl, optionally substituted carbocyclyl,         optionally substituted heterocyclyl, optionally substituted         aryl, optionally substituted heteroaryl, a nitrogen protecting         group, or a group of formula (i), (ii) or (iii);     -   each occurrence of R^(A1) is independently hydrogen, optionally         substituted alkyl, optionally substituted alkenyl, optionally         substituted alkynyl, optionally substituted carbocyclyl,         optionally substituted heterocyclyl, optionally substituted         aryl, optionally substituted heteroaryl, an oxygen protecting         group when attached to an oxygen atom, a sulfur protecting group         when attached to an sulfur atom, a nitrogen protecting group         when attached to a nitrogen atom, or two R^(A1) groups, together         with the nitrogen atom to which they are attached, are joined to         form an optionally substituted heterocyclic or optionally         substituted heteroaryl ring;     -   each instance of R² is independently hydrogen, optionally         substituted alkyl, optionally substituted alkenyl, optionally         substituted alkynyl, optionally substituted carbocyclyl,         optionally substituted heterocyclyl, optionally substituted         aryl, optionally substituted heteroaryl, a nitrogen protecting         group, or a group of the formula (i), (ii), or (iii);     -   Formulae (i), (ii), and (iii) are:

-   -   each instance of R′ is independently hydrogen or optionally         substituted alkyl;     -   X is O, S, or NR^(X);     -   R^(X) is hydrogen, optionally substituted alkyl, optionally         substituted alkenyl, optionally substituted alkynyl, optionally         substituted carbocyclyl, optionally substituted heterocyclyl,         optionally substituted aryl, optionally substituted heteroaryl,         or a nitrogen protecting group;     -   Y is O, S, or NR^(Y);     -   R^(Y) is hydrogen, optionally substituted alkyl, optionally         substituted alkenyl, optionally substituted alkynyl, optionally         substituted carbocyclyl, optionally substituted heterocyclyl,         optionally substituted aryl, optionally substituted heteroaryl,         or a nitrogen protecting group;     -   R^(P) is hydrogen, optionally substituted alkyl, optionally         substituted alkenyl, optionally substituted alkynyl, optionally         substituted carbocyclyl, optionally substituted heterocyclyl,         optionally substituted aryl, optionally substituted heteroaryl,         an oxygen protecting group when attached to an oxygen atom, a         sulfur protecting group when attached to a sulfur atom, or a         nitrogen protecting group when attached to a nitrogen atom;     -   R^(L) is optionally substituted C₁-50 alkyl, optionally         substituted C₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl,         optionally substituted heteroC₁₋₅₀ alkyl, optionally substituted         heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl,         or a polymer;     -   provided that at least one instance of R^(Q), R², R⁶, or R⁷ is a         group of the formula (i), (ii), or (iii).

In some embodiments, a cationic lipid in a provided composition or method is a compound of formula I. In some embodiments, a cationic lipid in a provided composition or method is a compound of formula I, wherein the compound comprises one or more basic groups. In some embodiments, a cationic lipid in a provided composition or method is a compound of formula I, wherein the compound comprises one or more amino groups.

In certain embodiments, a group of formula (i) represents a group of formula (i-a) or a group of formula (i-b):

wherein each variable is independently as defined above and described herein. In some embodiments, a group of formula (i) is a group of formula (i-a). In some embodiments, a group of formula (i) is a group of formula (i-b).

In some embodiments, at least one instance of R¹ is a group of formula (iv). In some embodiments, at least one instance of R¹ is a group of formula (iv), wherein at least one of R⁶ and R⁷ is a group of formula (i), (ii) or (iii). In some embodiments, at least one instance of R¹ is a group of formula (iv), wherein each of R⁶ and R⁷ is independently a group of formula (i), (ii) or (iii).

In some embodiments, each R¹ is independently a group of formula (iv). In some embodiments, each R¹ is independently a group of formula (iv), wherein at least one of R⁶ and R⁷ is a group of formula (i), (ii) or (iii). In some embodiments, each R¹ is independently a group of formula (iv), wherein each of R⁶ and R⁷ is independently a group of formula (i), (ii) or (iii). In some embodiments, each R¹ is independently a group of formula (iv), wherein each of R⁶ and R⁷ is independently a group of formula (i). In some embodiments, each R¹ is independently a group of formula (iv), wherein each of R⁶ and R⁷ is independently a group of formula (ii). In some embodiments, each R¹ is independently a group of formula (iv), wherein each of R⁶ and R⁷ is independently a group of formula (iii). In some embodiments, each R¹ is independently a group of formula (iv), wherein each of R⁶ and R⁷ is independently a group of formula (i-a). In some embodiments, each R¹ is independently a group of formula (iv), wherein each of R⁶ and R⁷ is independently a group of formula (i-b).

In some embodiments, each instance of R′ is hydrogen.

In some embodiments, L is an optionally substituted alkylene.

In some embodiments, a group of formula (iv) is of formula

wherein q is an integer between 1 and 50, inclusive, and each of R⁶ and R⁷ is independently as defined above and described herein.

As generally defined above, p is an integer of between 1 and 9, inclusive. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, p is 4. In certain embodiments, p is 5. In certain embodiments, p is 6. In certain embodiments, p is 7. In certain embodiments, p is 8. In certain embodiments, p is 9.

In some embodiments, p is 1. In some embodiments, a compound of formula I is a compound of formula (I-a):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, p is 2. In some embodiments, a compound of formula I is a compound of formula (I-p2):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, p is 3. In some embodiments, a compound of formula I is a compound of formula (I-p3):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, p is 4. In some embodiments, a compound of formula I is a compound of formula (I-p4):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, p is 5. In some embodiments, a compound of formula I is a compound of formula (I-p5):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, p is 6. In some embodiments, a compound of formula I is a compound of formula (I-p6):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, p is 7. In some embodiments, a compound of formula I is a compound of formula (I-p7):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, p is 8. In some embodiments, a compound of formula I is a compound of formula (I-p8):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, p is 9. In some embodiments, a compound of formula I is a compound of formula (I-p9):

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

As generally defined above, each instance of Q is independently O, S, or NR^(Q), wherein R^(Q) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of the formula (i), (ii), or (iii).

In certain embodiments, at least one instance of Q is O. In certain embodiments, each instance of Q is O. In certain embodiments, at least one instance of Q is S. In certain embodiments, each instance of Q is S. In certain embodiments, at least one instance of Q is NR^(Q), wherein R^(Q) is as defined above and described herein. In certain embodiments, each instance of Q is NR^(Q), wherein each R^(Q) is independently as defined above and described herein. In certain embodiments, each instance of R^(Q) is independently hydrogen or a group of the formula (i), (ii), or (iii).

As generally defined above, R^(Q) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of the formula (i), (ii) or (iii).

In some embodiments, R^(Q) is hydrogen. In some embodiments, R^(Q) is optionally substituted alkyl. In some embodiments, R^(Q) is optionally substituted alkenyl. In some embodiments, R^(Q) is optionally substituted alkynyl. In some embodiments, R^(Q) is carbocyclyl. In some embodiments, R^(Q) is optionally substituted heterocyclyl. In some embodiments, R^(Q) is optionally substituted aryl. In some embodiments, R^(Q) is optionally substituted heteroaryl. In some embodiments, R^(Q) is a nitrogen protecting group. In some embodiments, R^(Q) is a group of formula (i), (ii) or (iii). In some embodiments, R^(Q) is a group of formula (i). In some embodiments, R^(Q) is a group of formula (ii). In some embodiments, R^(Q) is a group of formula (iii).

As generally defined above, each instance of R¹ is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, halogen, —OR^(A1), —N(R^(A1))₂, or —SR^(A1), or a group of formula (iv), wherein each of R^(A1) and formula (iv) is independently as defined above and described herein.

In some embodiments, R¹ is hydrogen.

In certain embodiments, R¹ is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In certain embodiments, at least one instance of R¹ is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

In certain embodiments, R¹ is optionally substituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionally substituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionally substituted C₃₋₄alkyl. In certain embodiments, at least one instance of R¹ is optionally substituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionally substituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionally substituted C₃₋₄alkyl.

In certain embodiments, R¹ is optionally substituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl, optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl, or optionally substituted C₃₋₄alkenyl. In certain embodiments, at least one instance of R¹ is optionally substituted alkenyl, e.g., optionally substituted C₂₋₆ alkenyl, optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl, or optionally substituted C₃₋₄alkenyl.

In certain embodiments, R¹ is optionally substituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl, or optionally substituted C₃₋₄alkynyl. In certain embodiments, at least one instance of R¹ is optionally substituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl, or optionally substituted C₃₋₄alkynyl.

In certain embodiments, R¹ is optionally substituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl, optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆ carbocyclyl, optionally substituted C₅ carbocyclyl, or optionally substituted C₆ carbocyclyl. In certain embodiments, at least one instance of R¹ is optionally substituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl, optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆ carbocyclyl, optionally substituted C₅ carbocyclyl, or optionally substituted C₆ carbocyclyl.

In some embodiments, R¹ is optionally substituted heterocyclyl, e.g., optionally substituted 3-14 membered heterocyclyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted 5-8 membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl. In certain embodiments, at least one instance of R¹ is optionally substituted heterocyclyl, e.g., optionally substituted 3-14 membered heterocyclyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted 5-8 membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl.

In some embodiments, R¹ is optionally substituted aryl. In some embodiments, R¹ is optionally substituted phenyl. In some embodiments, R¹ is phenyl. In some embodiments, R¹ is substituted phenyl. In certain embodiments, at least one instance of R¹ is optionally substituted aryl, e.g., optionally substituted phenyl.

In some embodiments, R¹ is optionally substituted heteroaryl, e.g., optionally substituted 5-14 membered heteroaryl, optionally substituted 5-10 membered heteroaryl, optionally substituted 5-6 membered heteroaryl, optionally substituted 5 membered heteroaryl, or optionally substituted 6 membered heteroaryl. In certain embodiments, at least one instance of R¹ is optionally substituted heteroaryl, e.g., optionally substituted 5-14 membered heteroaryl, optionally substituted 5-10 membered heteroaryl, optionally substituted 5-6 membered heteroaryl, optionally substituted 5 membered heteroaryl, or optionally substituted 6 membered heteroaryl.

In some embodiments, R¹ is halogen. In some embodiments, R¹ is —F. In some embodiments, R¹ is —Cl. In some embodiments, R¹ is —Br. In some embodiments, R¹ is —I.

In some embodiments, R¹ is —OR^(A1), wherein R^(A1) is as defined above and described herein. In some embodiments, R¹ is —N(R^(A1))₂, wherein each R^(A1) is independently as defined above and described herein. In some embodiments, R¹ is —SR^(A1), wherein R^(A1) is as defined above and described herein.

In some embodiments, an R¹ alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted. In some embodiments, an R¹ alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted with an optionally substituted amino group. In some embodiments, an R¹ alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted with an optionally substituted hydroxyl group. In some embodiments, an R¹ alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted with an optionally substituted thiol group. In any of the above embodiments, an R¹ alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may be substituted, for example, with an optionally substituted amino group (e.g., —NR⁶R⁷), an optionally substituted hydroxyl group (e.g., —OR⁶), an optionally substituted thiol group (e.g., —SR⁶), or with a group of formula (i), (ii), or (iii), wherein each instance of R⁶ and R⁷ is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, and a sulfur protecting group when attached to a sulfur atom, or a group of formula (i), (ii), or (iii).

In some embodiments, R¹ is an optionally substituted natural amino acid side chain. In some embodiments, R¹ is a natural amino acid side chain. In some embodiments, R¹ is an optionally substituted unnatural amino acid side chain. In some embodiments, R¹ is an unnatural amino acid side chain.

In certain embodiments, each instance of R¹ is the same. In certain embodiments, at least one R¹ group is different. In certain embodiments, each R¹ group is different.

In certain embodiments, R¹ is an alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group substituted with an amino group of the formula —NR⁶R⁷.

In certain embodiments, R¹ is a group of formula (iv):

wherein: L is an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteralkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene, or combination thereof, and each of R⁶ and R⁷ is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of formula (i), (ii) or (iii):

wherein each of R′, Y, R^(P), R^(L) and X is independently as defined above and described herein.

In some embodiments, at least one instance of R¹ is an alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group substituted with an amino group of the formula —NR⁶R⁷. In some embodiments, at least one instance of R¹ is a group of formula (iv). In some embodiments, at least one instance of R¹ is a group of formula (iv), wherein at least one instance of R⁶ and R⁷ is a group of the formula (i), (ii) or (iii). In some embodiments, at least one instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (i), (ii) or (iii). In some embodiments, at least one instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (i). In some embodiments, at least one instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (ii). In some embodiments, at least one instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (iii).

In some embodiments, each instance of R¹ is a group of formula (iv). In some embodiments, each instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (i), (ii) or (iii). In some embodiments, each instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (i), (ii) or (iii). In some embodiments, each instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (i). In some embodiments, each instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (ii). In some embodiments, each instance of R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ is a group of the formula (iii).

In certain embodiments, at least two instances of R¹ is a group of formula (iv). In certain embodiments, at least three instances of R¹ is a group of formula (iv). In certain embodiments, at least four instances of R¹ is a group of formula (iv). In certain embodiments, at least five instances of R¹ is a group of formula (iv). In certain embodiments, at least six instances of R¹ is a group of formula (iv). In certain embodiments, at least seven instances of R¹ is a group of formula (iv). In certain embodiments, at least eight instances of R¹ is a group of formula (iv). In certain embodiments, at least nine instances of R¹ is a group of formula (iv). In certain embodiments, each instance of R¹ is a group of formula (iv).

In certain embodiments, L is an optionally substituted alkylene; e.g., optionally substituted C₁₋₅₀alkylene, optionally substituted C₁₋₄₀alkylene, optionally substituted C₁₋₃₀alkylene, optionally substituted C₁₋₂₀alkylene, optionally substituted C₄₋₂₀alkylene, optionally substituted C₆₋₂₀alkylene, optionally substituted C₈₋₂₀alkylene, optionally substituted C₁₀₋₂₀ alkylene, optionally substituted C₁₋₆alkylene, optionally substituted C₂₋₆alkylene, optionally substituted C₃₋₆alkylene, optionally substituted C₄₋₆alkylene, optionally substituted C₄₋₅alkylene, or optionally substituted C₃₋₄alkylene. In some embodiments, L is optionally substituted C₁ alkylene. In some embodiments, L is optionally substituted C₂ alkylene. In some embodiments, L is optionally substituted C₃ alkylene. In some embodiments, L is optionally substituted C₄ alkylene. In some embodiments, L is optionally substituted C₅ alkylene. In some embodiments, L is optionally substituted C₆ alkylene. In some embodiments, L is optionally substituted C₇ alkylene. In some embodiments, L is optionally substituted C₈ alkylene. In some embodiments, L is optionally substituted C₉ alkylene. In some embodiments, L is optionally substituted C₁₀ alkylene. In some embodiments, L is —CH₂—. In some embodiments, L is —(CH₂)₂—. In some embodiments, L is —(CH₂)₃—. In some embodiments, L is —(CH₂)₄—. In some embodiments, L is —(CH₂)₅—. In some embodiments, L is —(CH₂)₆—. In some embodiments, L is —(CH₂)₇—. In some embodiments, L is —(CH₂)₈—. In some embodiments, L is —(CH₂)₉—. In some embodiments, L is —(CH₂)₁₀—.

In certain embodiments, L is an optionally substituted alkenylene, e.g., optionally substituted C₂₋₅₀alkenylene, optionally substituted C₂₋₄₀alkenylene, optionally substituted C₂₋₃₀alkenylene, optionally substituted C₂₋₂₀alkenylene, optionally substituted C₄₋₂₀alkenylene, optionally substituted C₆₋₂₀alkenylene, optionally substituted C₈₋₂₀alkenylene, optionally substituted C₁₋₂₀alkenylene, optionally substituted C₂₋₆alkenylene, optionally substituted C₃₋₆ alkenylene, optionally substituted C₄₋₆alkenylene, optionally substituted C₄₋₅alkenylene, or optionally substituted C₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted alkynylene, e.g., optionally substituted C₂₋₅₀alkynylene, optionally substituted C₂₋₄₀alkynylene, optionally substituted C₂₋₃₀alkynylene, optionally substituted C₂₋₂₀alkynylene, optionally substituted C₄₋₂₀alkynylene, optionally substituted C₆₋₂₀alkynylene, optionally substituted C₈₋₂₀alkynylene, optionally substituted C₁₀₋₂₀ alkynylene, optionally substituted C₂₋₆alkynylene, optionally substituted C₃₋₆ alkynylene, optionally substituted C₄₋₆alkynylene, optionally substituted C₄₋₅alkynylene, or optionally substituted C₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted heteroalkylene; e.g., optionally substituted heteroC₁₋₅₀alkylene, optionally substituted heteroC₁₋₄₀alkylene, optionally substituted heteroC₁₋₃₀alkylene, optionally substituted heteroC₁₋₂₀alkylene, optionally substituted heteroC₄₋₂₀alkylene, optionally substituted heteroC₆₋₂₀alkylene, optionally substituted heteroC₃₋₂₀alkylene, optionally substituted heteroC₁₀₋₂₀alkylene, optionally substituted heteroC₁₋₆alkylene, optionally substituted heteroC₂₋₆alkylene, optionally substituted heteroC₃₋₆alkylene, optionally substituted heteroC₄₋₆alkylene, optionally substituted heteroC₄₋₅alkylene, or optionally substituted heteroC₃₋₄alkylene. In some embodiments, L is optionally substituted heteroC₂alkylene. In some embodiments, L is optionally substituted heteroC₃alkylene. In some embodiments, L is optionally substituted heteroC₄alkylene. In some embodiments, L is optionally substituted heteroC₅alkylene. In some embodiments, L is optionally substituted heteroC₆alkylene. In some embodiments, L is optionally substituted heteroC₇alkylene. In some embodiments, L is optionally substituted heteroC₈alkylene. In some embodiments, L is optionally substituted heteroC₉alkylene. In some embodiments, L is optionally substituted heteroC₁₀alkylene.

In certain embodiments, L is an optionally substituted heteroalkenylene, e.g., optionally substituted heteroC₂₋₅₀alkenylene, optionally substituted heteroC₂₋₄₀alkenylene, optionally substituted heteroC₂₋₃₀alkenylene, optionally substituted heteroC₂₋₂₀alkenylene, optionally substituted heteroC₄₋₂₀alkenylene, optionally substituted heteroC₆₋₂₀alkenylene, optionally substituted heteroC₈₋₂₀alkenylene, optionally substituted heteroC₁₀₋₂₀alkenylene, optionally substituted heteroC₂₋₆alkenylene, optionally substituted heteroC₃₋₆alkenylene, optionally substituted heteroC₄₋₆alkenylene, optionally substituted heteroC₄₋₅alkenylene, or optionally substituted heteroC₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted heteralkynylene, e.g., optionally substituted heteroC₂₋₅₀alkynylene, optionally substituted heteroC₂₋₄₀alkynylene, optionally substituted heteroC₂₋₃₀alkynylene, optionally substituted heteroC₂₋₂₀alkynylene, optionally substituted heteroC₄₋₂₀alkynylene, optionally substituted heteroC₆₋₂₀alkynylene, optionally substituted heteroC₈₋₂₀alkynylene, optionally substituted heteroC₁₀₋₂₀alkynylene, optionally substituted heteroC₂₋₆alkynylene, optionally substituted heteroC₃₋₆alkynylene, optionally substituted heteroC₄₋₆alkynylene, optionally substituted heteroC₄₋₅alkynylene, or optionally substituted heteroC₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted carbocyclylene, e.g., optionally substituted C₃₋₁₀carbocyclylene, optionally substituted C₅₋₈carbocyclylene, optionally substituted C₅₋₆carbocyclylene, optionally substituted C₅carbocyclylene, or optionally substituted C₆carbocyclylene.

In certain embodiments, L is an optionally substituted heterocyclylene, e.g., optionally substituted 3-14 membered heterocyclylene, optionally substituted 3-10 membered heterocyclylene, optionally substituted 5-8 membered heterocyclylene, optionally substituted 5-6 membered heterocyclylene, optionally substituted 5-membered heterocyclylene, or optionally substituted 6-membered heterocyclylene.

In certain embodiments, L is an optionally substituted arylene, e.g., optionally substituted phenylene. In some embodiments, L is optionally substituted phenylene. In some embodiments, L is substituted phenylene. In some embodiments, L is unsubstituted phenylene.

In certain embodiments, L is an optionally substituted heteroarylene, e.g., optionally substituted 5-14 membered heteroarylene, optionally substituted 5-10 membered heteroarylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 5-membered heteroarylene, or optionally substituted 6-membered heteroarylene.

In certain embodiments, wherein L is an optionally substituted alkylene group, the group of formula (iv) is a group of the formula

wherein q is an integer between 1 and 50, inclusive, and each of R⁶ and R⁷ is independently as defined above and described herein.

In certain embodiments, q is an integer between 1 and 40, inclusive. In certain embodiments, q is an integer between 1 and 30, inclusive. In certain embodiments, q is an integer between 1 and 20, inclusive. In certain embodiments, q is an integer between 1 and 10, inclusive. In certain embodiments, q is an integer between 4 and 20, inclusive. In certain embodiments, q is an integer between 6 and 20, inclusive. In certain embodiments, q is an integer between 2 and 10, inclusive. In certain embodiments, q is an integer between 2 and 9, inclusive. In certain embodiments, q is an integer between 2 and 8, inclusive. In certain embodiments, q is an integer between 2 and 7, inclusive. In certain embodiments, q is an integer between 2 and 6, inclusive. In certain embodiments, q is an integer between 2 and 5, inclusive. In certain embodiments, q is an integer between 2 and 4, inclusive. In certain embodiments, q is an integer between 3 and 10, inclusive. In certain embodiments, q is an integer between 3 and 8, inclusive. In certain embodiments, q is an integer between 3 and 7, inclusive. In certain embodiments, q is an integer between 3 and 6, inclusive. In certain embodiments, q is an integer between 3 and 5, inclusive. In certain embodiments, q is 3 or 4. In certain embodiments, q is an integer between 3 and 9, inclusive. In certain embodiments, q is an integer between 8 and 20, inclusive. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6. In certain embodiments, q is 7. In certain embodiments, q is 8. In certain embodiments, q is 9. In certain embodiments, q is 10.

As generally defined above, each R⁶ is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of formula (i), (ii) or (iii).

In some embodiments, R⁶ is hydrogen.

In some embodiments, R⁶ is optionally substituted alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₅₀ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₄₀ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₃₀ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₂₀ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₃ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₂ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₁ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₀ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₉ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₈ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₇ alkyl. In some embodiments, R⁶ is optionally substituted C₂₋₆ alkyl.

In some embodiments, R⁶ is optionally substituted C₄₋₅₀ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₄₀ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₃₀ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₂₀ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₃ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₂ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₁ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₀ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₉ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₈ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₇ alkyl. In some embodiments, R⁶ is optionally substituted C₄₋₆ alkyl.

In some embodiments, R⁶ is optionally substituted C₆₋₅₀ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₄₀ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₃₀ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₂₀ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₃ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₂ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₁ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₀ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₉ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₈ alkyl. In some embodiments, R⁶ is optionally substituted C₆₋₇ alkyl.

In some embodiments, R⁶ is optionally substituted C₈₋₅₀ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₄₀ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₃₀ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₂₀ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₃ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₂ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₁ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₀ alkyl. In some embodiments, R⁶ is optionally substituted C₈₋₉ alkyl.

In some embodiments, R⁶ is optionally substituted C₉50 alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₄₀ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₃₀ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₂₀ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₃ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₂ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₁ alkyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₀ alkyl.

In some embodiments, R⁶ is optionally substituted C₁₀₋₅₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₄₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₃₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₂₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₃ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₂ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₁ alkyl.

In some embodiments, R⁶ is optionally substituted C₁₁₋₅₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₄₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₃₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₂₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₃ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₂ alkyl.

In some embodiments, R⁶ is optionally substituted C₁₂₋₅₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₄₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₃₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₂₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₃ alkyl.

In some embodiments, R⁶ is optionally substituted C₆ alkyl. In some embodiments, R⁶ is optionally substituted C₇ alkyl. In some embodiments, R⁶ is optionally substituted C₈ alkyl. In some embodiments, R⁶ is optionally substituted C₉ alkyl. In some embodiments, R⁶ is optionally substituted C₁₀ alkyl. In some embodiments, R⁶ is optionally substituted C₁₁ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂ alkyl. In some embodiments, R⁶ is optionally substituted C₁₃ alkyl. In some embodiments, R⁶ is optionally substituted C₁₄ alkyl. In some embodiments, R⁶ is optionally substituted C₁₅ alkyl. In some embodiments, R⁶ is optionally substituted C₁₆ alkyl. In some embodiments, R⁶ is optionally substituted C₁₇ alkyl. In some embodiments, R⁶ is optionally substituted C₁₈ alkyl. In some embodiments, R⁶ is optionally substituted C₁₉ alkyl. In some embodiments, R⁶ is optionally substituted C₂₀ alkyl.

In some embodiments, for example, in any of the above embodiments, R⁶ is a substituted alkyl group. In some embodiments, R⁶ is an unsubstituted alkyl group. In some embodiments, R⁶ is an optionally substituted straight-chain alkyl group. In some embodiments, R⁶ is a substituted straight-chain alkyl group. In some embodiments, R⁶ is an unsubstituted straight-chain alkyl group. In some embodiments, R⁶ is an optionally substituted branched alkyl group. In some embodiments, R⁶ is a substituted branched alkyl group. In some embodiments, R⁶ is an unsubstituted branched alkyl group.

In some embodiments, R⁶ is optionally substituted alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₅₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₄₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₃₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₂₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₃ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₂ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₁ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₋₆ alkenyl.

In some embodiments, R⁶ is optionally substituted C₄₋₅₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₄₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₃₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₂₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₁ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₃ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₂ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₁ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₄₋₆ alkenyl.

In some embodiments, R⁶ is optionally substituted C₆₋₅₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₄₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₃₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₂₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₃ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₂ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₁ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₆₋₇ alkenyl.

In some embodiments, R⁶ is optionally substituted C₈₋₅₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₄₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₃₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₂₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₃ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₂ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₁ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₈₋₉ alkenyl.

In some embodiments, R⁶ is optionally substituted C₉₋₅₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₄₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₃₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₂₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₃ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₂ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₁ alkenyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₀ alkenyl.

In some embodiments, R⁶ is optionally substituted C₁₀₋₅₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₄₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₃₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₂₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₃ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₂ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₁ alkenyl.

In some embodiments, R⁶ is optionally substituted C₁₁₋₅₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₄₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₃₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₂₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₋₁₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₋₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₋₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₃ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₂ alkenyl.

In some embodiments, R⁶ is optionally substituted C₁₂₋₅₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₄₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₃₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₂₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₃ alkenyl.

In some embodiments, R⁶ is optionally substituted C₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₀ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₁ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₂ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₃ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₄ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₅ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₆ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₇ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₈ alkenyl. In some embodiments, R⁶ is optionally substituted C₁₉ alkenyl. In some embodiments, R⁶ is optionally substituted C₂₀ alkenyl.

In some embodiments, for example, in any of the above embodiments, R⁶ is a substituted alkenyl group. In some embodiments, R⁶ is an unsubstituted alkenyl group. In some embodiments, R⁶ is an optionally substituted straight-chain alkenyl group. In some embodiments, R⁶ is a substituted straight-chain alkenyl group. In some embodiments, R⁶ is an unsubstituted straight-chain alkenyl group. In some embodiments, R⁶ is an optionally substituted branched alkenyl group. In some embodiments, R⁶ is a substituted branched alkenyl group. In some embodiments, R⁶ is an unsubstituted branched alkenyl group.

In some embodiments, R⁶ is optionally substituted alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₅₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₄₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₃₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₂₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₃ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₂ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₁ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₁₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₋₆ alkynyl.

In some embodiments, R⁶ is optionally substituted C₄₋₅₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₄₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₃₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₂₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₁ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₃ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₂ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₁ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₁₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₄₋₆ alkynyl.

In some embodiments, R⁶ is optionally substituted C₆₋₅₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₄₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₃₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₂₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₃ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₂ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₁ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₁₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₆₋₇ alkynyl.

In some embodiments, R⁶ is optionally substituted C₈₋₅₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₄₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₃₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₂₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₃ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₂ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₁ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₁₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₈₋₉ alkynyl.

In some embodiments, R⁶ is optionally substituted C₉₋₅₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₄₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₃₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₂₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₃ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₂ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₁ alkynyl. In some embodiments, R⁶ is optionally substituted C₉₋₁₀ alkynyl.

In some embodiments, R⁶ is optionally substituted C₁₀₋₅₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₄₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₃₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₂₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₃ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₂ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀₋₁₁ alkynyl.

In some embodiments, R⁶ is optionally substituted C₁₁₋₅₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₄₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₋₃₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₂₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₃ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁₋₁₂ alkynyl.

In some embodiments, R⁶ is optionally substituted C₁₂₋₅₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₄₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₃₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₂₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂₋₁₃ alkynyl.

In some embodiments, R⁶ is optionally substituted C₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₀ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₁ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₂ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₃ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₄ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₅ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₆ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₇ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₈ alkynyl. In some embodiments, R⁶ is optionally substituted C₁₉ alkynyl. In some embodiments, R⁶ is optionally substituted C₂₀ alkynyl.

In some embodiments, for example, in any of the above embodiments, R⁶ is a substituted alkynyl group. In some embodiments, R⁶ is an unsubstituted alknyl group. In some embodiments, R⁶ is an optionally substituted straight-chain alkynyl group. In some embodiments, R⁶ is a substituted straight-chain alkynyl group. In some embodiments, R⁶ is an unsubstituted straight-chain alkynyl group. In some embodiments, R⁶ is an optionally substituted branched alkynyl group. In some embodiments, R⁶ is a substituted branched alkynyl group. In some embodiments, R⁶ is an unsubstituted branched alkynyl group.

In some embodiments, R⁶ is optionally substituted carbocyclyl. In some embodiments, R⁶ is optionally substituted heterocyclyl. In some embodiments, R⁶ is optionally substituted aryl. In some embodiments, R⁶ is optionally substituted heteroaryl. In some embodiments, R⁶ is a nitrogen protecting group.

In some embodiments, R⁶ is a group of formula (i). In some embodiments, R⁶ is a group of formula (i-a). In some embodiments, R⁶ is a group of formula (i-a1).

In some embodiments, R⁶ is a group of formula (i-b). In some embodiments, R is a group of formula (ii). In some embodiments, R⁶ is a group of formula (iii).

In some embodiments, R⁶ is substituted with one or more hydroxyl groups. In some embodiments, R⁶ is substituted with one hydroxyl group. In some embodiments, R⁶ is substituted with one 2-hydroxyl group (C1 is the carbon atom directly bonded to the nitrogen atom depicted in formula (iv)).

As generally defined above, each R⁷ is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of formula (i), (ii) or (iii).

In some embodiments, R⁷ is hydrogen.

In some embodiments, R⁷ is optionally substituted alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₅₀ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₄₀ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₃₀ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₂₀ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₃ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₂ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₁ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₀ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₉ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₈ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₇ alkyl. In some embodiments, R⁷ is optionally substituted C₂₋₆ alkyl.

In some embodiments, R⁷ is optionally substituted C₄₋₅₀ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₄₀ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₃₀ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₂₀ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₃ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₂ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₁ alkyl. In some embodiments, R⁷ is optionally substituted C₄-10 alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₉ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₈ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₇ alkyl. In some embodiments, R⁷ is optionally substituted C₄₋₆ alkyl.

In some embodiments, R⁷ is optionally substituted C₆₋₅₀ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₄₀ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₃₀ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₂₀ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₃ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₂ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₁ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₀ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₉ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₈ alkyl. In some embodiments, R⁷ is optionally substituted C₆₋₇ alkyl.

In some embodiments, R⁷ is optionally substituted C₈₋₅₀ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₄₀ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₃₀ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₂₀ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₃ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₂ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₁ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₀ alkyl. In some embodiments, R⁷ is optionally substituted C₈₋₉ alkyl.

In some embodiments, R⁷ is optionally substituted C₉₋₅₀ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₄₀ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₃₀ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₂₀ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₃ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₂ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₁ alkyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₀ alkyl.

In some embodiments, R⁷ is optionally substituted C₀₀-50 alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₄₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₃₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₂₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₀₀₋₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₃ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₂ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₁ alkyl.

In some embodiments, R⁷ is optionally substituted C₁₁₋₅₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₄₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₃₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₂₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₁₋₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₃ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₂ alkyl.

In some embodiments, R⁷ is optionally substituted C₁₂₋₅₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₄₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₃₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₂₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₃ alkyl.

In some embodiments, R⁷ is optionally substituted C₆ alkyl. In some embodiments, R⁷ is optionally substituted C₇ alkyl. In some embodiments, R⁷ is optionally substituted C₈ alkyl. In some embodiments, R⁷ is optionally substituted C₉ alkyl. In some embodiments, R⁷ is optionally substituted C₁₀ alkyl. In some embodiments, R⁷ is optionally substituted C₁₁ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂ alkyl. In some embodiments, R⁷ is optionally substituted C₁₃ alkyl. In some embodiments, R⁷ is optionally substituted C₁₄ alkyl. In some embodiments, R⁷ is optionally substituted C₁₅ alkyl. In some embodiments, R⁷ is optionally substituted C₁₆ alkyl. In some embodiments, R⁷ is optionally substituted C₁₇ alkyl. In some embodiments, R⁷ is optionally substituted C₁₈ alkyl. In some embodiments, R⁷ is optionally substituted C₁₉ alkyl. In some embodiments, R⁷ is optionally substituted C₂₀ alkyl.

In some embodiments, for example, in any of the above embodiments, R⁷ is a substituted alkyl group. In some embodiments, R⁷ is an unsubstituted alkyl group. In some embodiments, R⁷ is an optionally substituted straight-chain alkyl group. In some embodiments, R⁷ is a substituted straight-chain alkyl group. In some embodiments, R⁷ is an unsubstituted straight-chain alkyl group. In some embodiments, R⁷ is an optionally substituted branched alkyl group. In some embodiments, R⁷ is a substituted branched alkyl group. In some embodiments, R⁷ is an unsubstituted branched alkyl group.

In some embodiments, R⁷ is optionally substituted alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₅₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₄₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₃₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₂₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₃ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₂ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₁ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₋₆ alkenyl.

In some embodiments, R⁷ is optionally substituted C₄₋₅₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₄₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₃₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₂₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₃ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₂ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₁ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₄₋₆ alkenyl.

In some embodiments, R⁷ is optionally substituted C₆₋₅₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₄₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₃₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₂₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₃ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₂ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₁ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₆₋₇ alkenyl.

In some embodiments, R⁷ is optionally substituted C₈₋₅₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₄₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₃₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₂₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₃ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₂ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₁ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₈₋₉ alkenyl.

In some embodiments, R⁷ is optionally substituted C₉₋₅₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₄₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₃₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₂₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₃ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₂ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₁ alkenyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₀ alkenyl.

In some embodiments, R⁷ is optionally substituted C₁₀₋₅₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₄₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₃₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₂₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₃ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₂ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₁ alkenyl.

In some embodiments, R⁷ is optionally substituted C₁₁₋₅₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₄₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₃₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₂₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₃ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₂ alkenyl.

In some embodiments, R⁷ is optionally substituted C₁₂₋₅₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₄₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₃₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₂₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₃ alkenyl.

In some embodiments, R⁷ is optionally substituted C₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₈ alkenyl. In some embodiments, R⁷ is optionally substituted C₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₀ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₂ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₃ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₄ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₅ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₆ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₇ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₁ alkenyl. In some embodiments, R⁷ is optionally substituted C₁₉ alkenyl. In some embodiments, R⁷ is optionally substituted C₂₀ alkenyl.

In some embodiments, for example, in any of the above embodiments, R⁷ is a substituted alkenyl group. In some embodiments, R⁷ is an unsubstituted alkenyl group. In some embodiments, R⁷ is an optionally substituted straight-chain alkenyl group. In some embodiments, R⁷ is a substituted straight-chain alkenyl group. In some embodiments, R⁷ is an unsubstituted straight-chain alkenyl group. In some embodiments, R⁷ is an optionally substituted branched alkenyl group. In some embodiments, R⁷ is a substituted branched alkenyl group. In some embodiments, R⁷ is an unsubstituted branched alkenyl group.

In some embodiments, R⁷ is optionally substituted alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₅₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₄₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₃₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₂₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₃ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₂ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₁ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₁₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₋₆ alkynyl.

In some embodiments, R⁷ is optionally substituted C₄₋₅₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₄₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₃₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₂₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₃ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₂ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₁ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₁₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₄₋₆ alkynyl.

In some embodiments, R⁷ is optionally substituted C₆₋₅₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₄₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₃₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₂₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₃ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₂ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₁ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₁₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₆₋₇ alkynyl.

In some embodiments, R⁷ is optionally substituted C₈₋₅₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₄₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₃₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₂₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₅₋₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₃ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₂ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₁ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₁₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₈₋₉ alkynyl.

In some embodiments, R⁷ is optionally substituted C₉50 alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₄₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₃₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₂₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₃ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₂ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₁ alkynyl. In some embodiments, R⁷ is optionally substituted C₉₋₁₀ alkynyl.

In some embodiments, R⁷ is optionally substituted C₁₀₋₅₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₄₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₃₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₂₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₃ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₂ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀₋₁₁ alkynyl.

In some embodiments, R⁷ is optionally substituted C₁₁₋₅₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₄₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₃₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₂₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₋₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₃ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁₋₁₂ alkynyl.

In some embodiments, R⁷ is optionally substituted C₁₂₋₅₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₄₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₃₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₂₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂₋₁₃ alkynyl.

In some embodiments, R⁷ is optionally substituted C₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₀ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₁ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₂ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₃ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₄ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₅ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₆ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₇ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₈ alkynyl. In some embodiments, R⁷ is optionally substituted C₁₉ alkynyl. In some embodiments, R⁷ is optionally substituted C₂₀ alkynyl.

In some embodiments, for example, in any of the above embodiments, R⁷ is a substituted alkynyl group. In some embodiments, R⁷ is an unsubstituted alkynyl group. In some embodiments, R⁷ is an optionally substituted straight-chain alkynyl group. In some embodiments, R⁷ is a substituted straight-chain alkynyl group. In some embodiments, R⁷ is an unsubstituted straight-chain alkynyl group. In some embodiments, R⁷ is an optionally substituted branched alkynyl group. In some embodiments, R⁷ is a substituted branched alkynyl group. In some embodiments, R⁷ is an unsubstituted branched alkynyl group.

In some embodiments, R⁷ is optionally substituted carbocyclyl. In some embodiments, R⁷ is optionally substituted heterocyclyl. In some embodiments, R⁷ is optionally substituted aryl. In some embodiments, R⁷ is optionally substituted heteroaryl. In some embodiments, R⁷ is a nitrogen protecting group.

In some embodiments, R⁷ is a group of formula (i). In some embodiments, R⁷ is a

group of formula (i-a). In some embodiments, R⁷ is a group of formula (i-a1). In some embodiments, R⁷ is a group of formula (i-b). In some embodiments, R⁷ is a group of formula (ii). In some embodiments, R⁷ is a group of formula (iii).

In some embodiments, at least one instance of R⁶ and R⁷ is a group of the formula (i), (ii) or (iii). In some embodiments, each instance of R⁶ and R⁷ is independently a group of the formula (i), (ii) or (iii). In some embodiments, each instance of R⁶ and R⁷ is independently a group of the formula (i). In some embodiments, each instance of R⁶ and R⁷ is independently a group of the formula (i-a). In some embodiments, each instance of R⁶ and R⁷ is independently a group of the formula (i-b). In some embodiments, each instance of R⁶ and R⁷ is independently a group of the formula (ii). In some embodiments, each instance of R⁶ and R⁷ is independently a group of the formula (iii).

In some embodiments, R⁶ and R⁷ are the same. In some embodiments, R⁶ and R⁷ are different.

In certain embodiments, both R⁶ and R⁷ are hydrogen. In certain embodiments, R⁶ is hydrogen and R⁷ is a group of the formula (i), (ii), or (iii). In certain embodiments, R⁶ is hydrogen and R⁷ is a group of the formula (i). In certain embodiments, R⁶ is hydrogen and R⁷ is a group of the formula (ii). In certain embodiments, R⁶ is hydrogen and R⁷ is a group of the formula (iii). In certain embodiments, each of R⁶ and R⁷ is independently a group of the formula (i), (ii), or (iii). In certain embodiments, each of R⁶ and R⁷ is independently a group of the formula (i). In certain embodiments, each of R⁶ and R⁷ is independently a group of the formula (ii). In certain embodiments, each of R⁶ and R⁷ is independently a group of the formula (iii). In certain embodiments, R⁶ and R⁷ are the same group, which is selected from formulas (i), (ii), and (iii). In some embodiments, R⁶ and R⁷ are the same group of formula (i). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a1). In some embodiments, R⁶ and R⁷ are the same group of formula (i-b).

In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is as defined above and described herein. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substituted C₂₋₅₀alkenyl, optionally substituted C₂₋₅₀alkynyl, optionally substituted heteroC₁₋₅₀alkyl, optionally substituted heteroC₂₋₅₀alkenyl, or optionally substituted heteroC₂₋₅₀alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₅₀alkyl, optionally substituted C₅₋₅₀alkenyl, optionally substituted C₅₋₅₀alkynyl, optionally substituted heteroC₅₋₅₀alkyl, optionally substituted heteroC₅₋₅₀alkenyl, or optionally substituted heteroC₅₋₅₀alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₄₀alkyl, optionally substituted C₅₋₄₀alkenyl, optionally substituted C₅₋₄₀alkynyl, optionally substituted heteroC₅₋₄₀alkyl, optionally substituted heteroC₅₋₄₀alkenyl, or optionally substituted heteroC₅₋₄₀alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₃₀alkyl, optionally substituted C₅₋₃₀alkenyl, optionally substituted C₅₋₃₀alkynyl, optionally substituted heteroC₅₋₃₀alkyl, optionally substituted heteroC₅₋₃₀ alkenyl, or optionally substituted heteroC₅₋₃₀alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₂₅alkyl, optionally substituted C₅₋₂₅alkenyl, optionally substituted C₅₋₂₅alkynyl, optionally substituted heteroC₅₋₂₅alkyl, optionally substituted heteroC₅₋₂₅alkenyl, or optionally substituted heteroC₅₋₂₅alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₂₀alkyl, optionally substituted C₅₋₂₀alkenyl, optionally substituted C₅₋₂₀alkynyl, optionally substituted heteroC₅₋₂₀alkyl, optionally substituted heteroC₅₋₂₀alkenyl, or optionally substituted heteroC₅₋₂₀alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₁₅alkyl, optionally substituted C₅₋₁₅alkenyl, optionally substituted C₅₋₁₅alkynyl, optionally substituted heteroC₅₋₁₅alkyl, optionally substituted heteroC₅₋₁₅alkenyl, or optionally substituted heteroC₅₋₁₅alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅ alkyl, optionally substituted C₅ alkenyl, optionally substituted C₅ alkynyl, optionally substituted heteroC₅ alkyl, optionally substituted heteroC₅ alkenyl, or optionally substituted heteroC₅ alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₆ alkyl, optionally substituted C₆ alkenyl, optionally substituted C₆ alkynyl, optionally substituted heteroC₆alkyl, optionally substituted heteroC₆alkenyl, or optionally substituted heteroC₆alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₇ alkyl, optionally substituted C₇ alkenyl, optionally substituted C₇ alkynyl, optionally substituted heteroC₇alkyl, optionally substituted heteroC₇alkenyl, or optionally substituted heteroC₇alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₈ alkyl, optionally substituted C₈ alkenyl, optionally substituted C₈ alkynyl, optionally substituted heteroC₈alkyl, optionally substituted heteroC₈alkenyl, or optionally substituted heteroC₈alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₉ alkyl, optionally substituted C₉ alkenyl, optionally substituted C₉ alkynyl, optionally substituted heteroC₉alkyl, optionally substituted heteroC₉alkenyl, or optionally substituted heteroC₉alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₀ alkyl, optionally substituted C₁₀ alkenyl, optionally substituted C₁₀ alkynyl, optionally substituted heteroC₁₀alkyl, optionally substituted heteroC₁₀alkenyl, or optionally substituted heteroC₁₀alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₁ alkyl, optionally substituted C₁₁ alkenyl, optionally substituted C₁₁ alkynyl, optionally substituted heteroC₁₁alkyl, optionally substituted heteroC₁₁alkenyl, or optionally substituted heteroC₁₁alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₂ alkyl, optionally substituted C₁₂ alkenyl, optionally substituted C₁₂ alkynyl, optionally substituted heteroC₁₂alkyl, optionally substituted heteroC₁₂alkenyl, or optionally substituted heteroC₁₂alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₃ alkyl, optionally substituted C₁₃ alkenyl, optionally substituted C₁₃ alkynyl, optionally substituted heteroC₁₃alkyl, optionally substituted heteroC₁₃alkenyl, or optionally substituted heteroC₁₃alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₄ alkyl, optionally substituted C₁₄ alkenyl, optionally substituted C₁₄ alkynyl, optionally substituted heteroC₁₄alkyl, optionally substituted heteroC₁₄alkenyl, or optionally substituted heteroC₁₄alkynyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₅ alkyl, optionally substituted C₁₅ alkenyl, optionally substituted C₁₅ alkynyl, optionally substituted heteroC₁₅alkyl, optionally substituted heteroC₁₅alkenyl, or optionally substituted heteroC₁₅alkynyl.

In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is as defined above and described herein. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₋₅₀ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₅₀alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₄₀alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₃₀alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₂₅alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₂₀alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₁₅alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₆ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₇ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₈ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₉ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₀ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₁ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₂ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₃ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₄ alkyl. In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₅ alkyl.

As generally defined above, each occurrence of R^(A1) is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to an sulfur atom, a nitrogen protecting group when attached to a nitrogen atom, or two R^(A1) groups, together with the nitrogen atom to which they are attached, are joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring.

In some embodiments, R^(A1) is hydrogen. In some embodiments, R^(A1) is optionally substituted alkyl. In some embodiments, R^(A1) is optionally substituted alkenyl. In some embodiments, R^(A1) is optionally substituted alkynyl. In some embodiments, R^(A1) is optionally substituted carbocyclyl. In some embodiments, R^(A1) is optionally substituted heterocyclyl. In some embodiments, R^(A1) is optionally substituted aryl. In some embodiments, R^(A1) is optionally substituted heteroaryl. In some embodiments, R^(A1) is an oxygen protecting group when attached to an oxygen atom. In some embodiments, R^(A1) is a sulfur protecting group when attached to a sulfur atom. In some embodiments, R^(A1) is a nitrogen protecting group when attached to a nitrogen atom. In some embodiments, two R^(A1) groups, together with the nitrogen atom to which they are attached, are joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring.

As generally defined above, each instance of R² is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group, or a group of the formula (i), (ii), or (iii):

wherein each of R′, Y, R^(P), R^(L) and X is independently as defined above and described herein.

In some embodiments, R² is hydrogen. In some embodiments, at least one instance of R² is hydrogen. In some embodiments, each instance of R² is hydrogen.

In certain embodiments, R² is optionally substituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionally substituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionally substituted C₃₋₄alkyl. In certain embodiments, at least one instance of R² is optionally substituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionally substituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionally substituted C₃₋₄alkyl.

In certain embodiments, R² is optionally substituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl, optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl, or optionally substituted C₃₋₄alkenyl. In certain embodiments, at least one instance of R² is optionally substituted alkenyl, e.g., optionally substituted C₂₋₆alkenyl, optionally substituted C₃₋₆alkenyl, optionally substituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl, or optionally substituted C₃₋₄alkenyl.

In certain embodiments, R² is optionally substituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl, or optionally substituted C₃₋₄alkynyl. In certain embodiments, at least one instance of R² is optionally substituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl, optionally substituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl, or optionally substituted C₃₋₄alkynyl.

In certain embodiments, R² is optionally substituted carbocyclyl, e.g., optionally substituted C₃₋₁₀carbocyclyl, optionally substituted C₅₋₈carbocyclyl, optionally substituted C₅₋₆ carbocyclyl, optionally substituted C₅ carbocyclyl, or optionally substituted C₆ carbocyclyl. In certain embodiments, at least one instance of R² is optionally substituted carbocyclyl, e.g., optionally substituted C₃₋₁₀carbocyclyl, optionally substituted C₅₋₈carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionally substituted C₆ carbocyclyl.

In certain embodiments, R² is optionally substituted heterocyclyl, e.g., optionally substituted 3-14 membered heterocyclyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted 5-8 membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl. In certain embodiments, at least one instance of R² is optionally substituted heterocyclyl, e.g., optionally substituted 3-14 membered heterocyclyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted 5-8 membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl.

In certain embodiments, R² is optionally substituted aryl, e.g., optionally substituted phenyl. In some embodiments, R² is optionally substituted phenyl. In some embodiments, R² is substituted phenyl. In some embodiments, R² is unsubstituted phenyl. In certain embodiments, at least one instance of R² is optionally substituted aryl, e.g., optionally substituted phenyl. In some embodiments, at least one instance of R² is optionally substituted phenyl. In some embodiments, at least one instance of R² is substituted phenyl. In some embodiments, at least one instance of R² is unsubstituted phenyl.

In certain embodiments, R² is optionally substituted heteroaryl, e.g., optionally substituted 5-14 membered heteroaryl, optionally substituted 5-10 membered heteroaryl, optionally substituted 5-6 membered heteroaryl, optionally substituted 5-membered heteroaryl, or optionally substituted 6-membered heteroaryl. In certain embodiments, at least one instance of R² is optionally substituted heteroaryl, e.g., optionally substituted 5-14 membered heteroaryl, optionally substituted 5-10 membered heteroaryl, optionally substituted 5-6 membered heteroaryl, optionally substituted 5-membered heteroaryl, or optionally substituted 6-membered heteroaryl.

In some embodiments, R² is a nitrogen protecting group. In some embodiments, at least one R² is a nitrogen protecting group.

In certain embodiments, R² is a group of the formula (i). In certain embodiments, R² is a group of the formula (ii). In certain embodiments, R² is a group of the formula (iii). In certain embodiments, at least one instance of R² is a group of the formula (i). In certain embodiments, at least one instance of R² is a group of the formula (ii). In certain embodiments, at least one instance of R² is a group of the formula (iii).

In certain embodiments, each instance of R² is a group other than formula (i), (ii), or (iii); in that instance, it follows that at least one R^(Q) is a group of the formula (i), (ii), or (iii), or at least one R¹ is a group of formula (iv), and at least one of R⁶ or R⁷ encompassed by R¹ is a group of the formula (i), (ii), or (iii). For example, in certain embodiments, both instances of R² are hydrogen, and thus at least one R^(Q) is a group of the formula (i), (ii), or (iii), or at least one R¹ is a group of formula (iv), and at least one of R⁶ or R⁷ encompassed by R¹ is a group of the formula (i), (ii), or (iii).

As generally defined above, each instance of R′ is independently hydrogen or optionally substituted alkyl. In some embodiments, R′ is hydrogen. In some embodiments, R′ is substituted alkyl. In certain embodiments, at least one instance of R′ is hydrogen. In certain embodiments, at least two instances of R′ is hydrogen. In certain embodiments, each instance of R′ is hydrogen. In certain embodiments, at least one instance of R′ is optionally substituted alkyl, e.g., methyl. In certain embodiments, at least two instances of R′ is optionally substituted alkyl, e.g., methyl. In some embodiments, at least one instance of R′ is hydrogen, and at least one instance of R′ is optionally substituted alkyl. In certain embodiments, one instance of R′ is optionally substituted alkyl, and the rest are hydrogen.

As generally defined above, X is O, S, or NR^(X). In some embodiments, X is O. In some embodiments, X is S. In some embodiments, X is NR^(X), wherein R^(X) is as defined above and described herein.

As generally defined above, R^(X) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group. In some embodiments, R^(X) is hydrogen. In some embodiments, R^(X) is optionally substituted alkyl. In some embodiments, R^(X) is optionally substituted alkenyl. In some embodiments, R^(X) is optionally substituted alkynyl. In some embodiments, R^(X) is optionally substituted carbocyclyl. In some embodiments, R^(X) is optionally substituted heterocyclyl. In some embodiments, R^(X) is optionally substituted aryl. In some embodiments, R^(X) is optionally substituted heteroaryl. In some embodiments, R^(X) is a nitrogen protecting group.

As generally defined above, Y is O, S, or NR^(Y). In some embodiments, Y is O. In some embodiments, Y is S. In some embodiments, Y is NR^(Y), wherein R^(Y) is as defined above and described herein.

As generally defined above, R^(Y) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group. In some embodiments, R^(Y) is hydrogen. In some embodiments, R^(Y) is optionally substituted alkyl. In some embodiments, R^(Y) is optionally substituted alkenyl. In some embodiments, R^(Y) is optionally substituted alkynyl. In some embodiments, R^(Y) is is optionally substituted carbocyclyl. In some embodiments, R^(Y) is optionally substituted heterocyclyl. In some embodiments, R^(Y) is optionally substituted aryl. In some embodiments, R^(Y) is is optionally substituted heteroaryl. In some embodiments, R^(Y) is a nitrogen protecting group.

As generally defined above, R^(P) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom. In some embodiments, R^(P) is hydrogen. In some embodiments, R^(P) is optionally substituted alkyl. In some embodiments, R^(P) is optionally substituted alkenyl. In some embodiments, R^(P) is optionally substituted alkynyl. In some embodiments, R^(P) is optionally substituted carbocyclyl. In some embodiments, R^(P) is optionally substituted heterocyclyl. In some embodiments, R^(P) is optionally substituted aryl. In some embodiments, R^(P) is optionally substituted heteroaryl. In some embodiments, R^(P) is an oxygen protecting group when attached to an oxygen atom. In some embodiments, R¹ is a sulfur protecting group when attached to a sulfur atom. In some embodiments, R¹ is a nitrogen protecting group when attached to a nitrogen atom.

As generally defined above, R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substituted C₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionally substituted heteroC₁₅₋₅₀ alkyl, optionally substituted heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.

In some embodiments, R^(L) is optionally substituted C₁₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₄₀ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₃₀ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₂₀ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₃ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₂ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₁ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₀ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₉ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₈ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₇ alkyl. In some embodiments, R^(L) is optionally substituted C₂₋₆ alkyl.

In some embodiments, R^(L) is optionally substituted C₄₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₄₀ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₃₀ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₂₀ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₃ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₂ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₀ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₀alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₉ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₈ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₇ alkyl. In some embodiments, R^(L) is optionally substituted C₄₋₆ alkyl.

In some embodiments, R^(L) is optionally substituted C₆₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₄₀ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₃₀ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₂₀ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₃ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₂ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₁ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₀ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₉ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₈ alkyl. In some embodiments, R^(L) is optionally substituted C₆₋₇ alkyl.

In some embodiments, R^(L) is optionally substituted C₈₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₄₀ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₃₀ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₂₀ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₃ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₂ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₁ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₀ alkyl. In some embodiments, R^(L) is optionally substituted C₈₋₉ alkyl.

In some embodiments, R^(L) is optionally substituted C₉₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₄₀ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₃₀ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₂₀ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₃ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₂ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₁ alkyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₀ alkyl.

In some embodiments, R^(L) is optionally substituted C₁₀₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₄₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₃₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₂₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₃ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₂ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₁ alkyl.

In some embodiments, R^(L) is optionally substituted C₁₁-50 alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₄₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₃₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₂₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₃ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₂ alkyl.

In some embodiments, R^(L) is optionally substituted C₁₂₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₄₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₃₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₂₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₃ alkyl.

In some embodiments, R^(L) is optionally substituted C₆ alkyl. In some embodiments, R^(L) is optionally substituted C₇ alkyl. In some embodiments, R^(L) is optionally substituted C₈ alkyl. In some embodiments, R^(L) is optionally substituted C₉ alkyl. In some embodiments, R^(L) is optionally substituted C₁₀ alkyl. In some embodiments, R^(L) is optionally substituted C₁₁ alkyl. In some embodiments, R^(L) is optionally substituted C₁₂ alkyl. In some embodiments, R^(L) is optionally substituted C₁₃ alkyl. In some embodiments, R^(L) is optionally substituted C₁₄ alkyl. In some embodiments, R^(L) is optionally substituted C₁₅ alkyl. In some embodiments, R^(L) is optionally substituted C₁₆ alkyl. In some embodiments, R^(L) is optionally substituted C₁₇ alkyl. In some embodiments, R^(L) is optionally substituted C₁₈ alkyl. In some embodiments, R^(L) is optionally substituted C₁₉ alkyl. In some embodiments, R^(L) is optionally substituted C₂₀ alkyl.

In some embodiments, for example, in any of the above embodiments, R^(L) is a substituted alkyl group. In some embodiments, R^(L) is an unsubstituted alkyl group. In some embodiments, R^(L) is an optionally substituted straight-chain alkyl group. In some embodiments, R^(L) is a substituted straight-chain alkyl group. In some embodiments, R^(L) is an unsubstituted straight-chain alkyl group. In some embodiments, R^(L) is an optionally substituted branched alkyl group. In some embodiments, R^(L) is a substituted branched alkyl group. In some embodiments, R^(L) is an unsubstituted branched alkyl group.

In certain embodiments, at least one instance of R^(L) is an unsubstituted alkyl. Exemplary unsubstituted alkyl groups include, but are not limited to, —CH₃, —C₂H₅, —C₃H₇, —C₄H₉, —C₅H₁₁, —C₆H₁₃, —C₇H₁₅, —C₈H₁₇, —C₉H₁₉, —C₁₀H₂₁, —C₁₁H₂₃, —C₁₂H₂₅, —C₁₃H₂₇, —C₁₄H₂₉, —C₁₅H₃₁, —C₁₆H₃₃, —C₁₇H₃₅, —C₁₈H₃₇, —C₁₉H₃₉, —C₂₀H₄₁, —C₂₁H₄₃, —C₂₂H₄₅, —C₂₃H₄₇, —C₂₄H₄₉, and —C₂₅H₅₁.

In certain embodiments, at least one instance of R^(L) is a substituted alkyl. For example, in certain embodimenets, at least one instance of R^(L) is an alkyl substituted with one or more fluorine substituents. Exemplary fluorinated alkyl groups include, but are not limited to:

In some embodiments, R^(L) is optionally substituted C₂₋₅₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₃₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₂₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₃ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₂ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₁ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₋₆ alkenyl.

In some embodiments, R^(L) is optionally substituted C₄₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₃₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₂₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₃ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₂ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₁ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₄₋₆ alkenyl.

In some embodiments, R^(L) is optionally substituted C₆₋₅₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₃₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₂₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₃ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₂ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₁ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₆₋₇ alkenyl.

In some embodiments, R^(L) is optionally substituted C₈₋₅₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₃₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₂₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₃ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₂ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₁ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₈₋₉ alkenyl.

In some embodiments, R^(L) is optionally substituted C₉₋₅₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₃₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₂₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₃ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₂ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₁ alkenyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₀ alkenyl.

In some embodiments, R^(L) is optionally substituted C₁₀₋₅₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₃₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₂₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₃ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₂ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₁ alkenyl.

In some embodiments, R^(L) is optionally substituted C₁₁₋₅₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₃₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₂₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₋₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₋₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₃ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₂ alkenyl.

In some embodiments, R^(L) is optionally substituted C₁₂₋₅₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₄₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₃₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₂₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₃ alkenyl.

In some embodiments, R^(L) is optionally substituted C₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₀ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₁ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₂ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₃ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₄ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₅ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₆ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₇ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₈ alkenyl. In some embodiments, R^(L) is optionally substituted C₁₉ alkenyl. In some embodiments, R^(L) is optionally substituted C₂₀ alkenyl.

In some embodiments, for example, in any of the above embodiments, R^(L) is a substituted alkyl group. In some embodiments, R^(L) is an unsubstituted alkyl group. In some embodiments, R^(L) is an optionally substituted straight-chain alkenyl group. In some embodiments, R^(L) is a substituted straight-chain alkenyl group. In some embodiments, R^(L) is an unsubstituted straight-chain alkenyl group. In some embodiments, R^(L) is an optionally substituted branched alkenyl group. In some embodiments, R^(L) is a substituted branched alkenyl group. In some embodiments, R^(L) is an unsubstituted branched alkenyl group.

Exemplary unsubstituted alkenyl group include, but are not limited to:

-   Myristoleic —(CH₂C)₇CH═CH(CH₂)₃CH₃, -   Palmitoliec —(CH₂)₇CH═CH(CH₂)₅CH₃, -   Sapienic —(CH₂)₄CH═CH(CH₂)₈CH₃, -   Oleic —(CH₂)₇CH═CH(CH₂)₇CH₃, -   Linoleic —(CH₂)₇CH═CHCH₂CH═CH(CH₂)₄CH₃, -   α-Linolenic —(CH₂)₇CH═CHCH₂CH═CHCH₂CH═CHCH₂CH₃, -   Arachinodonic —(CH₂)₃CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₄CH₃, -   Eicosapentaenoic —(CH₂)₃CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH₃, -   Erucic —(CH₂)₁₁CH═CH(CH₂)₇CH₃, and -   Docosahexaenoic— -   (CH₂)₂CH═CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CH—CH₂CH₃.

In some embodiments, wherein R^(L) is defined as a C₆₋₅₀alkyl or C₆₋₅₀alkenyl groups, such groups are meant to encompass lipophilic groups (also referred to as a “lipid tail”). Lipophilic groups comprise a group of molecules that include fats, waxes, oils, fatty acids, and the like. Lipid tails present in these lipid groups can be saturated and unsaturated, depending on whether or not the lipid tail comprises double bonds. The lipid tail can also comprise different lengths, often categorized as medium (i.e., with tails between 7-12 carbons, e.g., C₇₋₁₂ alkyl or C₇₋₁₂ alkenyl), long (i.e., with tails greater than 12 carbons and up to 22 carbons, e.g., C₁₃₋₂₂alkyl or C₁₃₋₂₂ alkenyl), or very long (i.e., with tails greater than 22 carbons, e.g., C₂₃₋₃₀ alkyl or C₂₃₋₃₀ alkenyl).

In some embodiments, R^(L) is optionally substituted C₂₋₅₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₄₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₃₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₂₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₃ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₂ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₁ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₁₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₋₆ alkynyl.

In some embodiments, R^(L) is optionally substituted C₄₋₅₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₄₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₃₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₂₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₃ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₂ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₁ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₁₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₄₋₆ alkynyl.

In some embodiments, R^(L) is optionally substituted C₆₋₅₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₄₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₃₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₂₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₃ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₂ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₁ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₁₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₆₋₇ alkynyl.

In some embodiments, R^(L) is optionally substituted C₈₋₅₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₄₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₃₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₂₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₃ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₂ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₁ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₁₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₈₋₉ alkynyl.

In some embodiments, R^(L) is optionally substituted C₉₋₅₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₄₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₃₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₂₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₃ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₂ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₁ alkynyl. In some embodiments, R^(L) is optionally substituted C₉₋₁₀ alkynyl.

In some embodiments, R^(L) is optionally substituted C₁₀₋₅₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₄₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₃₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₂₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₃ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₂ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀₋₁₁ alkynyl.

In some embodiments, R^(L) is optionally substituted C₁₁₋₅₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₄₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₃₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₂₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₃ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁₋₁₂ alkynyl.

In some embodiments, R^(L) is optionally substituted C₁₂₋₅₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₄₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₃₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₂₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂₋₁₃ alkynyl.

In some embodiments, R^(L) is optionally substituted C₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₀ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₁ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₂ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₃ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₄ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₅ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₆ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₇ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₈ alkynyl. In some embodiments, R^(L) is optionally substituted C₁₉ alkynyl. In some embodiments, R^(L) is optionally substituted C₂₀ alkynyl.

In some embodiments, for example, in any of the above embodiments, R^(L) is a substituted alkynyl group. In some embodiments, R^(L) is an unsubstituted alkynyl group. In some embodiments, R^(L) is an optionally substituted straight-chain alkyl group. In some embodiments, R^(L) is an optionally substituted straight-chain alkynyl group. In some embodiments, R^(L) is a substituted straight-chain alkynyl group. In some embodiments, R^(L) is an unsubstituted straight-chain alkynyl group. In some embodiments, R^(L) is an optionally substituted branched alkynyl group. In some embodiments, R^(L) is a substituted branched alkynyl group. In some embodiments, R^(L) is an unsubstituted branched alkynyl group.

In some embodiments, R^(L) is optionally substituted heteroC₁₋₅₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₅₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₃₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₂₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₃alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₂alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₁alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₆alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₄₋₅₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₃₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₂₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₃alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₂alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₁alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₆alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₆₋₅₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₃₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₂₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₃alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₂alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₁alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₇alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₈₋₅₀ alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₃₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₂₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₃alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₂alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₁alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₉alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₉₋₅₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₃₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₂₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₃alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₂alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₁alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₀alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₅₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₃₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₂₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₃alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₂alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₁alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₅₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₃₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₂₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₃alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₂alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₅₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₄₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₃₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₂₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₃alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₃alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₄alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₅alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₆alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₇alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₈alkyl. In some embodiments, R^(L) is optionally substituted heteroC₁₉alkyl. In some embodiments, R^(L) is optionally substituted heteroC₂₀alkyl.

In some embodiments, for example, in any of the above embodiments, R^(L) is a substituted heteroalkyl group. In some embodiments, R^(L) is an unsubstituted heteroalkyl group. In some embodiments, R^(L) is an optionally substituted straight-chain heteroalkyl group. In some embodiments, R^(L) is a substituted straight-chain heteroalkyl group. In some embodiments, R^(L) is an unsubstituted straight-chain heteroalkyl group. In some embodiments, R^(L) is an optionally substituted branched heteroalkyl group. In some embodiments, R^(L) is a substituted branched heteroalkyl group. In some embodiments, R^(L) is an unsubstituted branched heteroalkyl group.

Exemplary unsubstituted heteroalkyl groups include, but are not limited to:

In some embodiments, R^(L) is optionally substituted heteroC₂₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₄₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₃₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₂₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₃alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₂alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₁alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₆alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₄₋₅₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₄₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₃₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₂₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₃alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₂alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₁alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₆alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₆₋₅₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₄₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₃₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₂₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₃alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₂alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₁alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₇alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₈₋₅₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₄₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₃₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₂₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₅₋₁₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₃alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₂alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₉alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₉₋₅₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₄₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₃₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₂₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₃alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₂alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₁alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₀alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₅₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₄₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₃₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₂₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₃alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₂alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₁ alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₅₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₄₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₃₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₂₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₃alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₂alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₅₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₄₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₃₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₂₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₃alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₃alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₄alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₅alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₆alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₇alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₈alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₁₉alkenyl. In some embodiments, R^(L) is optionally substituted heteroC₂₀alkenyl.

In some embodiments, for example, in any of the above embodiments, R^(L) is a substituted heteroalkenyl group. In some embodiments, R^(L) is an unsubstituted heteroalkenyl group. In some embodiments, R^(L) is an optionally substituted straight-chain heteroalkenyl group. In some embodiments, R^(L) is a substituted straight-chain heteroalkenyl group. In some embodiments, R^(L) is an unsubstituted straight-chain heteroalkenyl group. In some embodiments, R^(L) is an optionally substituted branched heteroalkenyl group. In some embodiments, R^(L) is a substituted branched heteroalkenyl group. In some embodiments, R^(L) is an unsubstituted branched heteroalkenyl group.

In some embodiments, R^(L) is optionally substituted heteroC₂₋₅₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₄₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₃₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₂₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₃alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₂alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₁alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₋₆alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₄₋₅₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₄₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₃₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₂₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₃alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₂alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₁alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₄₋₆alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₆₋₅₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₄₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₃₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₂₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₃alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₂alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₁alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₆₋₇alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₈₋₅₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₄₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₃₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₂₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₃alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₂alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₁alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈₋₉alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₉₋₅₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₄₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₃₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₂₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₃alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₂alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₁alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₀alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₅₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₄₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₃₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₂₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₃alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₂alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₁alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₅₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₄₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₃₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₂₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₋₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₃alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₂alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₅₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₄₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₃₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₂₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₃alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₀alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₁alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₂alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₃alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₄alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₅alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₆alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₇alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₈alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₁₉alkynyl. In some embodiments, R^(L) is optionally substituted heteroC₂₀alkynyl.

In some embodiments, for example, in any of the above embodiments, R^(L) is a substituted heteroalkynyl group. In some embodiments, R^(L) is an unsubstituted heteroalkynyl group. In some embodiments, R^(L) is an optionally substituted straight-chain heteroalkynyl group. In some embodiments, R^(L) is a substituted straight-chain heteroalkynyl group. In some embodiments, R^(L) is an unsubstituted straight-chain heteroalkynyl group. In some embodiments, R^(L) is an optionally substituted branched heteroalkynyl group. In some embodiments, R^(L) is a substituted branched heteroalkynyl group. In some embodiments, R^(L) is an unsubstituted branched heteroalkynyl group.

In some embodiments, R^(L) is a polymer. As used herein, a “polymer”, in some embodiments, refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units. The polymer is in certain embodiments biocompatible (i.e., non-toxic). Exemplary polymers include, but are not limited to, cellulose polymers (e.g., hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, methylc cellulose, hydroxypropylmethylcellulose (HPMC)), dextran polymers, polymaleic acid polymers, poly(acrylic acid) polymers, poly(vinylalcohol) polymers, polyvinylpyrrolidone (PVP) polymers, and polyethyleneglycol (PEG) polymers, and combinations thereof.

In some embodiments, R^(L) is a lipophilic, hydrophobic and/or non-polar group. In some embodiments, R^(L) is a lipophilic group. In some embodiments, R^(L) is a hydrophobic group. In some embodiments, R^(L) is a non-polar group.

In some embodiments, when an R^(L) group is depicted as bisecting a carbon-carbon bond, e.g., of the formula (i), it is understood that R^(L) may be bonded to either carbon.

In some embodiments, at least one instance of R^(Q), R², R⁶, or R⁷ is a group of the formula (i), (ii), or (iii). In some embodiments, at least one instance of R⁶ or R⁷ of R¹ is a group of formula (i), (ii) or (iii). In some embodiments, at least one instance of R⁶ or R⁷ of R¹ is a group of formula (i). In some embodiments, at least one instance of R⁶ or R⁷ of R¹ is a group of formula (i-a). In some embodiments, at least one instance of R⁶ or R⁷ of R¹ is a group of formula (i-a1). In some embodiments, at least one instance of R⁶ or R⁷ of R¹ is a group of formula (i-b). In some embodiments, at least one instance of R⁶ or R⁷ of R¹ is a group of formula (ii). In some embodiments, at least one instance of R⁶ or R⁷ of R¹ is a group of formula (iii).

Various combinations of the above embodiments of Formula I are contemplated herein.

In some embodiments, wherein each instance of Q is O, the compound of formula I is a compound of formula I-a:

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In certain embodiments, at least one R¹ is a group of formula (iv). In certain embodiments, each instance of R¹ is a group of formula (iv). In certain embodiments, each instance of R² is independently hydrogen or optionally substituted C₁₋₆alkyl. In certain embodiments, each instance of R² is hydrogen. In certain embodiments, at least one instance of R² is a group of formula (i). In certain embodiments, at least one instance of R² is a group of formula (ii). In certain embodiments, at least one instance of R² is a group of formula (iii). In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3.

In some embodiments, wherein at least one R¹ is a group of formula (iv), a compound of formula I is a compound of formula I-b:

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In certain embodiments, each instance of R¹ is a group of formula (iv). In certain embodiments, each instance of R² is independently hydrogen or optionally substituted C₁₋₆alkyl. In certain embodiments, each instance of R² is hydrogen. In certain embodiments, at least one instance of R² is a group of formula (i). In certain embodiments, at least one instance of R² is a group of formula (ii). In certain embodiments, at least one instance of R² is a group of formula (iii). In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, L is an optionally substituted alkylene. In certain embodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ is a group of formula (ii). In certain embodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷ is a group of formula (i). In certain embodiments, R⁷ is a group of formula (ii). In certain embodiments, R⁷ is a group of formula (iii). In certain embodiments, both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii).

In some embodiments, wherein each instance of R¹ is a group the formula (iv), a compound of Formula I is a compound of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In certain embodiments, each instance of R² is independently hydrogen or optionally substituted C₁₆alkyl. In certain embodiments, each instance of R² is hydrogen. In certain embodiments, at least one instance of R² is a group of formula (i). In certain embodiments, at least one instance of R² is a group of formula (ii). In certain embodiments, at least one instance of R² is a group of formula (iii). In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, L is an optionally substituted alkylene. In certain embodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ is a group of formula (ii). In certain embodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷ is a group of formula (i). In certain embodiments, R⁷ is a group of formula (ii). In certain embodiments, R⁷ is a group of formula (iii). In certain embodiments, both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii).

In some embodiments, p=1. In some embodiments, a compound of formula I-c is a compound of formula I-c1:

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In certain embodiments, each instance of R² is independently hydrogen or optionally substituted C₁₋₆alkyl. In certain embodiments, each instance of R² is hydrogen. In certain embodiments, at least one instance of R² is a group of formula (i). In certain embodiments, at least one instance of R² is a group of formula (ii). In certain embodiments, at least one instance of R² is a group of formula (iii). In certain embodiments, L is an optionally substituted alkylene. In certain embodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ is a group of formula (ii). In certain embodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷ is a group of formula (i). In certain embodiments, R⁷ is a group of formula (ii). In certain embodiments, R⁷ is a group of formula (iii). In certain embodiments, both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii). In some embodiments, R⁶ and R⁷ are the same group of formula (i). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a1). In some embodiments, R⁶ and R⁷ are the same group of formula (i-b). In some embodiments, R⁶ and R⁷ are the same group of formula (ii). In some embodiments, R⁶ and R⁷ are the same group of formula (iii).

In some embodiments, each instance of R² is hydrogen. In some embodiments, a compound of formula I-c is a compound of formula I-c2:

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In certain embodiments, L is an optionally substituted alkylene. In certain embodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ is a group of formula (ii). In certain embodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷ is a group of formula (i). In certain embodiments, R⁷ is a group of formula (ii). In certain embodiments, R⁷ is a group of formula (iii). In certain embodiments, both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii). In some embodiments, R⁶ and R⁷ are the same group of formula (i). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a1). In some embodiments, R⁶ and R⁷ are the same group of formula (i-b). In some embodiments, R⁶ and R⁷ are the same group of formula (ii). In some embodiments, R⁶ and R⁷ are the same group of formula (iii).

In some embodiments, L is an optionally substituted alkylene. In some embodiments, a compound of formula I-c is a compound of formula I-c3:

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, q is an integer between 1 and 10, inclusive. In certain embodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ is a group of formula (ii). In certain embodiments, R⁶ is a group of formula (iii). In certain embodiments, R⁷ is a group of formula (i). In certain embodiments, R⁷ is a group of formula (ii). In certain embodiments, R⁷ is a group of formula (iii). In certain embodiments, both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii). In some embodiments, R⁶ and R⁷ are the same group of formula (i). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a1). In some embodiments, R⁶ and R⁷ are the same group of formula (i-b). In some embodiments, R⁶ and R⁷ are the same group of formula (ii). In some embodiments, R⁶ and R⁷ are the same group of formula (iii).

In some embodiments, a compound of formula I is a compound of formula I-d:

wherein each ofp, R² and R^(L) is independently as defined above and described herein.

In some embodiments, a compound of formula I is a compound of formula I-e:

wherein each of R² and R^(L) is independently as defined above and described herein.

In some embodiments, a compound of formula I is a compound of formula I-f:

wherein each of R² and R^(L) is independently as defined above and described herein.

In some embodiments, provided liposomes include a cationic lipid described in WO 2013063468 and in U.S. provisional application entitled “Lipid Formulations for Delivery of Messenger RNA” filed concurrently with the present application on even date, both of which are incorporated by reference herein. In some embodiments, a compound of formula I is a compound of formula I-c1-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each R² independently is hydrogen or C₁₋₃ alkyl;     -   each q independently is 2 to 6;     -   each R′ independently is hydrogen or C₁₋₃ alkyl;

and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² independently is hydrogen, methyl or ethyl. In some embodiments, each R² independently is hydrogen or methyl. In some embodiments, each R² is hydrogen.

In some embodiments, each q independently is 3 to 6. In some embodiments, each q independently is 3 to 5. In some embodiments, each q is 4.

In some embodiments, each R′ independently is hydrogen, methyl or ethyl. In some embodiments, each R′ independently is hydrogen or methyl. In some embodiments, each R′ independently is hydrogen.

In some embodiments, each R^(L) independently is C₈₋₁₂ alkyl. In some embodiments, each R^(L) independently is n-C₈₋₁₂ alkyl. In some embodiments, each R^(L) independently is C₉₋₁₁ alkyl. In some embodiments, each R^(L) independently is n-C₉₋₁₁ alkyl. In some embodiments, each R^(L) independently is C₁₀ alkyl. In some embodiments, each R^(L) independently is n-C₁₀ alkyl.

In some embodiments, each R² independently is hydrogen or methyl; each q independently is 3 to 5; each R′ independently is hydrogen or methyl; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q independently is 3 to 5; each R′ is hydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q is 4; each R′ is hydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, a compound of formula I is a compound of formula I-g:

wherein each of R^(L) is independently as defined above and described herein.

In some embodiments, a compound of formula I is a compound of formula X:

or a pharmaceutically acceptable salt thereof, wherein each variable is independently as defined above and described herein.

In some embodiments, a compound of formula I is a compound of formula X-1:

or a pharmaceutically acceptable salt thereof, wherein each R² is independently as defined above and described herein.

In some embodiments, a compound of formula I is

or a pharmaceutically acceptable salt thereof.

Additional examples of cationic lipids suitable for the present invention are described in WO 2013063468, which is incorporated by reference herein in its entirety.

Chemical Definitions

Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). The invention additionally contemplates compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example “C1-6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.

As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms (“C1-50 alkyl”). In some embodiments, an alkyl group has 1 to 40 carbon atoms (“C1-40 alkyl”). In some embodiments, an alkyl group has 1 to 30 carbon atoms (“C1-30 alkyl”). In some embodiments, an alkyl group has 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include, without limitation, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents. In certain embodiments, the alkyl group is an unsubstituted C1-50 alkyl. In certain embodiments, the alkyl group is a substituted C1-50 alkyl.

As used herein, “heteroalkyl” refers to an alkyl group as defined herein which further includes at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 50 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-50 alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 40 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-40 alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 30 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-30 alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-20 alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-10 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-9 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-8 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-7 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-6 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC1-5 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC1-4 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC1-3 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC1-2 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC1 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC2-6 alkyl”). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC1-50 alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC1-50 alkyl.

As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C2-50 alkenyl”). In some embodiments, an alkenyl group has 2 to 40 carbon atoms (“C2-40 alkenyl”). In some embodiments, an alkenyl group has 2 to 30 carbon atoms (“C2-30 alkenyl”). In some embodiments, an alkenyl group has 2 to 20 carbon atoms (“C2-20 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-4 alkenyl groups include, without limitation, ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C2-50 alkenyl. In certain embodiments, the alkenyl group is a substituted C2-50 alkenyl.

As used herein, “heteroalkenyl” refers to an alkenyl group as defined herein which further includes at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 50 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-50 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 40 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-40 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 30 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-30 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-20 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-10 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-9 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-8 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-7 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC2-6 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC2-5 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms. at least one double bond, and for 2 heteroatoms within the parent chain (“heteroC2-4 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC2-3 alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double, bond, and 1 or 2 heteroatoms within the parent chain (“heteroC2-6 alkenyl”). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC2-50 alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC2-50 alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C2-50 alkynyl”). An alkynyl group that has one or more triple bonds and one or more double bonds is also referred to as an “ene-yne”. In some embodiments, an alkynyl group has 2 to 40 carbon atoms (“C2-40 alkynyl”). In some embodiments, an alkynyl group has 2 to 30 carbon atoms (“C2-30 alkynyl”). In some embodiments, an alkynyl group has 2 to 20 carbon atoms (“C2-20 alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C2-50 alkynyl. In certain embodiments, the alkynyl group is a substituted C2-50 alkynyl.

As used herein, “heteroalkynyl” refers to an alkynyl group as defined herein which further includes at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 50 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-50 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 40 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-40 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 30 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-30 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-20 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-10 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-9 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-8 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-7 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC2-6 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC2-5 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and for 2 heteroatoms within the parent chain (“heteroC2-4 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC2-3 alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond. and 1 or 2 heteroatoms within the parent chain (“heteroC2-6 alkynyl”). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents. In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC2-50 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC2-50 alkynyl.

As used herein, “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C3-7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”). Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-8 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C3-10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-10 carbocyclyl.

In some embodiments, “carbocyclyl” or “carbocyclic” is referred to as a “cycloalkyl”, i.e., a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6, cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C4-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C3-10 cycloalkyl.

As used herein, “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“3-14 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)). and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance ofheterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation. tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

As used herein, “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C6-14 aryl. In certain embodiments, the aryl group is a substituted C6-14 aryl.

As used herein, “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation. pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.

As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups (e.g., aryl or heteroaryl moieties) as herein defined.

As used herein, the term “saturated” refers to a ring moiety that does not contain a double or triple bond, i.e., the ring contains all single bonds.

Affixing the suffix “-ene” to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl,

alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteralkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.

As understood from the above, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are, in certain embodiments, optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or. “unsubstituted” heteroalkynyl. “substituted” or “unsubstituted” carbocyclyl. “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORaa, —ON(Rbb)2, —N(Rbb)2, —N(Rbb)3+X—, —N(ORcc)Rbb, —SeH, —SeRaa, —SH, —SRaa, —SSRcc, —C(═O)Raa, —CO2H, —CHO, —C(ORcc)2, —CO2Raa, —OC(═O)Raa, —OCO2Raa, —C(═O)N(Rbb)2, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —OC(═NRbb)Raa, —OC(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —OC(═NRbb)N(Rbb)2, —NRbbC(═NRbb)N(Rbb)2, —C(═O)NRbbSO2Raa, —NRbbSO2Raa, —SO2N(Rbb)2, —SO2Raa, —SO2ORaa, —OSO2Raa, —S(═O)Raa, —OS(═O)Raa, —Si(Raa)3 —OSi(Raa)3 —C(═S)N(Rbb)2, —C(═O)SRaa, —C(═S)SRaa, —SC(═S)SRaa, —SC(═O)SRaa, —OC(═O)SRaa, —SC(═O)ORaa, —SC(═O)Raa, —P(═O)2Raa, —OP(═O)2Raa, —P(═O)(Raa)2, —OP(═O)(Raa)2, —OP(═O)(ORcc)2, —P(═O)2N(Rbb)2, —OP(═O)2N(Rbb)2, —P(═O)(NRbb)2, —OP(═O)(NRbb)2, —NRbbP(═O)(ORcc)2, —NRbbP(═O)(NRbb)2, —P(Rcc)2, —P(Rcc)3, —OP(Rcc)2, —OP(Rcc)₃, —B(Raa)2, —B(ORcc)2, —BRaa(ORcc), C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-14 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

or two geminal hydrogens on a carbon atom are replaced with the group ═O, ═S, ═NN(Rbb)₂, =NNRbbC(═O)Raa, =NNRbbC(═O)ORaa, =NNRbbS(═O)₂Raa, =NRbb, or =NORcc;

each instance of Raa is, independently, selected from C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Raa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

each instance of Rbb is, independently, selected from hydrogen, —OH, —ORaa, —N(Rcc)₂, —CN, —C(═O)Raa, —C(═O)N(Rcc)₂, —CO2Raa, —SO2Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)₂, —SO2N(Rcc)₂, —SO2Rcc, —SO20Rcc, —SORaa, —C(═S)N(Rcc)₂, —C(═O)SRcc, —C(═S)SRcc, —P(═O)₂Raa, —P(═O)(Raa)₂, —P(═O)₂N(Rcc)₂, —P(═O)(NRcc)₂, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rbb groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

each instance of Rcc is, independently, selected from hydrogen, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

each instance of Rdd is, independently, selected from halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORee, —ON(Rff)₂, —N(Rff)₂, —N(Rff)₃+X—, —N(ORee)Rff, —SH, —SRee, —SSRee, —C(═O)Ree, —CO2H, —CO2Ree, —OC(═O)Ree, —OCO2Ree, —C(═O)N(Rff)₂, —OC(═O)N(Rff)₂, —NRffC(═O)Ree, —NRffCO2Ree, —NRffC(═O)N(Rff)₂, —C(═NRff)ORee, —OC(═NRff)Ree, —OC(═NRff)ORee, —C(═NRff)N(Rff)₂, —OC(═NRff)N(Rff)₂, —NRffC(═NRff)N(Rff)₂, —NRffSO2Ree, —S 02N(Rff)₂, —SO2Ree, —SO2ORee, —OSO2Ree, —S(═O)Ree, —Si(Ree)₃, —OSi(Ree)₃, —C(═S)N(Rff)₂, —C(═O)SRee, —C(═S)SRee, —SC(═S)SRee, —P(═O)₂Ree, —P(═O)(Ree)₂, —OP(═O)(Ree)₂, —OP(═O)(ORee)₂, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups, or two geminal Rdd substituents can be joined to form ═O or ═S;

each instance of Ree is, independently, selected from C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;

each instance of Rff is, independently, selected from hydrogen, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl and 5-10 membered heteroaryl, or two Rff groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; and

each instance of Rgg is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —OC1-50 alkyl, —ON(C1-50 alkyl)₂, —N(C1-50 alkyl)₂, —N(C1-50 alkyl)₃+X—, —NH(C1-50 alkyl)₂+X—, —NH2(C1-50 alkyl)+X—, —NH3+X—, —N(OC1-50 alkyl)(C1-50 alkyl), —N(OH)(C1-50 alkyl), —NH(OH), —SH, —SC1-50 alkyl, —SS(C1-50 alkyl), —C(═O)(C1-50 alkyl), —CO2H, —CO2(C1-50 alkyl), —OC(═O)(C1-50 alkyl), —OCO2(C1-50 alkyl), —C(═O)NH2, —C(═O)N(C1-50 alkyl)₂, —OC(═O)NH(C1-50 alkyl), —NHC(═O)(C1-50 alkyl), —N(C1-50 alkyl)C(═O)(C1-50 alkyl), —NHCO2(C1-50 alkyl), —NHC(═O)N(C1-50 alkyl)₂, —NHC(═O)NH(C1-50 alkyl), —NHC(═O)NH2, —C(═NH)O(C1-50 alkyl), —OC(═NH)(C1-50 alkyl), —OC(═NH)OC1-50 alkyl, —C(═NH)N(C1-50 alkyl)₂, —C(═NH)NH(C1-50 alkyl), —C(═NH)NH2, —OC(═NH)N(C1-50alky 1)₂, —OC(NH)NH(C1-50 alkyl), —OC(NH)NH2, —NHC(NH)N(C1-50 alkyl)₂, —NHC(═NH)NH2, —NHSO2 (C1-50 alkyl), —SO2N (C1-50 alkyl)₂, —SO2NH (C1-50 alkyl), —SO2NH2, —SO2C1-50 alkyl, —SO2OC1-50 alkyl, —OSO2C1-6 alkyl, —SOC1-6 alkyl, —Si(C1-50 alkyl)3, —OSi(C1-6 alkyl)3 —C(═S)N(C1-50 alkyl)2, C(═S)NH(C1-50 alkyl), C(═S)NH2, —C(═O)S(C1-6 alkyl), —C(═S)SC1-6 alkyl, —SC(═S)SC1-6 alkyl, —P(═O)2(C1-50 alkyl), —P(═O)(C1-50 alkyl)2, —OP(═O)(C1-50 alkyl)2, —OP(═O)(OC1-50 alkyl)2, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal Rgg substituents can be joined to form ═O or ═S;

wherein X— is a counterion.

As used herein, the term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

As used herein, a “counterion” is a negatively charged group associated with a positively charged quarternary amine in order to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F—, Cl—, Br—, I—), NO3-, C1O4-, OH—, H2PO4-, HSO4-, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like).

Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary nitrogen atom substitutents include, but are not limited to, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRbb)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)2N(Rcc)2, —P(═O)(NRcc)2, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, together with the N atom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.

Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary nitrogen atom substitutents include, but are not limited to, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRbb)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)2N(Rcc)2, —P(═O)(NRcc)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, together with the nitrogen atom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.

In certain embodiments, the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups include, but are not limited to, —OH, —ORaa, —N(Rcc)2, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRcc)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g., —C(═O)Raa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g., —C(═O)ORaa) include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methyl sulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g., —S(═O)₂Raa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), j3-trimethyl silylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacyl derivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethyl silyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups include, but are not limited to, —Raa, —N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, Si(Raa)₃, —P(Rcc)2, —P(Rcc)₃, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2, —P(═O)2N(Rbb)2, and —P(═O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a, 4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethyl silylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picoly1 N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

In certain embodiments, the substituent present on an sulfur atom is an sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups include, but are not limited to, —Raa, —N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, C(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3, —P(Rcc)2, —P(Rcc)3, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2, —P(═O)2N(Rbb)2, and —P(═O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

As used herein, a “leaving group” is an art-understood term referring to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage, wherein the molecular fragment is an anion or neutral molecule. See, for example, Smith, March's Advanced Organic Chemistry 6th ed. (501-502). Exemplary leaving groups include, but are not limited to, halo (e.g., chloro, bromo, iodo) and sulfonyl substituted hydroxyl groups (e.g., tosyl, mesyl, besyl).

Other Definitions

As used herein, use of the phrase “at least one instance” refers to one instance, but also encompasses more than one instance, e.g., for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 instances, and up to 100 instances.

As used herein, a “polymer” refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units.

“Attached” refers to the covalent attachment of a group.

As used herein, “lipophilic” refers to the ability of a group to dissolve in fats, oils, lipids, and lipophilic non-polar solvents such as hexane or toluene. In general, a lipophilic group refers to an unsubstituted n-alkyl or unsubstituted n-alkenyl group having 6 to 50 carbon atoms, e.g., 6 to 40, 6 to 30, 6 to 20, 8 to 20, 8 to 19, 8 to 18, 8 to 17, 8 to 16, or 8 to 15 carbon atoms.

As used herein, the term “salt” or “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate. digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium. quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.

Second or Additional Cationic Lipids

In some embodiments, liposomes may comprise a second or additional cationic lipid. As used herein, the phrase “cationic lipid” refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO 2010/053572 (and particularly, C12-200 described at paragraph [00225]) and WO 2012/170930, both of which are incorporated herein by reference. In certain embodiments, the compositions and methods of the invention employ a lipid nanoparticles comprising an ionizable cationic lipid described in U.S. provisional patent application 61/617,468, filed Mar. 29, 2012 (incorporated herein by reference), such as, e.g, (15Z, 18Z)—N,N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z, 18Z)—N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)—N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-5, 15, 18-trien-1-amine (HGT5002).

In some embodiments, the second or additional cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA” is used. (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium or “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S. Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propane or “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”. Additional exemplary cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”, 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”, 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”, 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”, N-dioleyl-N,N-dimethylammonium chloride or “DODAC”, N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or “DMRIE”, 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane or “CLinDMA”, 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy 1-1-(cis,cis-9′, 1-2′-octadecadienoxy)propane or “CpLinDMA”, N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”, 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”, 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”, 1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”, 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-DMA”, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or “DLin-K-XTC2-DMA”, and 2-(2,2-di((9Z, 12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.

In some embodiments, the second or additional cationic lipid may be chosen from XTC (2,2-Dilinoley 1-4-dimethylaminoethy 1-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3 aR, 5 s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011/068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61/617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (Provisional Patent Application No. 61/617,468), aminoalcohol lipidoids such as those disclosed in WO2010/053572, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids” J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al. “Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materials for low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).

Non-Cationic/Helper Lipids

In some embodiments, provided liposomes contain one or more non-cationic (“helper”) lipids. As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected H, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof.

In some embodiments, such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids. In some embodiments, the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10% to about 70% of the total lipid present in a liposome. In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

Cholesterol-Based Lipids

In some embodiments, provided liposomes comprise one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in a liposome. In some embodiments, The percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 5, %, 10%, greater than 20%, greater than 30%, or greater than 40%.

PEGylated Lipids

In some embodiments, provided liposomes comprise one or more PEGylated lipids. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of the cationic and, in some embodiments, other lipids together which comprise the liposome. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C₆-C₂₀ length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).

In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C₁₄ or C₁₈). The PEG-modified phospholipid and derivitized lipids of the present invention may comprise a molar ratio from about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome.

According to various embodiments, the selection of second or additional cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly. In some embodiments, the percentage of PEG-modified lipid in a liposome may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 15%.

Polymer

In some embodiments, a suitable liposome according to the present invention further includes a polymer, in combination with one or more cationic lipids as described and, in some embodiments, other carriers including various lipids described herein. Thus, in some embodiments, liposomal delivery vehicles, as used herein, also encompass polymer containing nanoparticles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI). When PEI is present, it may be branched PEI of a molecular weight ranging from 10 to 40 kDA, e.g., 25 kDa branched PEI (Sigma #408727).

In some embodiments, a suitable liposome formulation contains a combination of one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids one or more PEG-modified lipids, and/or one or more polymers. As a non-limiting example, a suitable liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid may be between about 30-50:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:32:25:3, respectively. mRNA

The present invention can be used to deliver any mRNA. mRNA is typically thought of as the type of RNA that carries information from DNA to the ribosome. The existence of mRNA is usually very brief and includes processing and translation, followed by degradation. Typically, in eukaryotic organisms, mRNA processing comprises the addition of a “cap” on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end. A typical cap is a 7-methylguanosine cap, which is a guanosine that is linked through a 5′-5′-triphosphate bond to the first transcribed nucleotide. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3′ end of the mRNA molecule. The presence of this “tail” serves to protect the mRNA from exonuclease degradation. Messenger RNA typically is translated by the ribosomes into a series of amino acids that make up a protein.

Any mRNA capable of being translated into one or more peptides (e.g., proteins) or peptide fragments is contemplated as within the scope of the present invention. In some embodiments, an mRNA encodes one or more naturally occurring peptides. In some embodiments, an mRNA encodes one or more modified or non-natural peptides.

In some embodiments an mRNA encodes an intracellular protein. In some embodiments, an mRNA encodes a cytosolic protein. In some embodiments, an mRNA encodes a protein associated with the actin cytoskeleton. In some embodiments, an mRNA encodes a protein associated with the plasma membrane. In some specific embodiments, an mRNA encodes a transmembrane protein. In some specific embodiments an mRNA encodes an ion channel protein. In some embodiments, an mRNA encodes a perinuclear protein. In some embodiments, an mRNA encodes a nuclear protein. In some specific embodiments, an mRNA encodes a transcription factor. In some embodiments, an mRNA encodes a chaperone protein. In some embodiments, an mRNA encodes an intracellular enzyme (e.g., mRNA encoding an enzyme associated with urea cycle or lysosomal storage metabolic disorders). In some embodiments, an mRNA encodes a protein involved in cellular metabolism, DNA repair, transcription and/or translation. In some embodiments, an mRNA encodes an extracellular protein. In some embodiments, an mRNA encodes a protein associated with the extracellular matrix. In some embodiments an mRNA encodes a secreted protein. In specific embodiments, an mRNA used in the composition and methods of the invention may be used to express functional proteins or enzymes that are excreted or secreted by one or more target cells into the surrounding extracellular fluid (e.g., mRNA encoding hormones and/or neurotransmitters).

In some embodiments, the compositions and methods of the invention provide for delivery of mRNA encoding a secreted protein. In some embodiments, the compositions and methods of the invention provide for delivery of mRNA encoding one or more secreted proteins listed in Table 1; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein.

TABLE 1 Secreted Proteins Uniprot ID Protein Name Gene Name A1E959 Odontogenic ameloblast-associated protein ODAM A1KZ92 Peroxidasin-like protein PXDNL A1L453 Serine protease 38 PRSS38 A1L4H1 Soluble scavenger receptor cysteine-rich domain- SSC5D containing protein SSC5D A2RUU4 Colipase-like protein 1 CLPSL1 A2VDF0 Fucose mutarotase FUOM A2VEC9 SCO-spondin SSPO A3KMH1 von Willebrand factor A domain-containing VWA8 protein 8 A4D0S4 Laminin subunit beta-4 LAMB4 A4D1T9 Probable inactive serine protease 37 PRSS37 A5D8T8 C-type lectin domain family 18 member A CLEC18A A6NC86 phospholipase A2 inhibitor and Ly6/PLAUR PINLYP domain-containing protein A6NCI4 von Willebrand factor A domain-containing VWA3A protein 3A A6ND01 Probable folate receptor delta FOLR4 A6NDD2 Beta-defensin 108B-like A6NE02 BTB/POZ domain-containing protein 17 BTBD17 A6NEF6 Growth hormone 1 GH1 A6NF02 NPIP-like protein LOC730153 A6NFB4 HCG1749481, isoform CRA_k CSH1 A6NFZ4 Protein FAM24A FAM24A A6NG13 Glycosyltransferase 54 domain-containing protein A6NGN9 IgLON family member 5 IGLON5 A6NHN0 Otolin-1 OTOL1 A6NHN6 Nuclear pore complex-interacting protein-like 2 NPIPL2 A6NI73 Leukocyte immunoglobulin-like receptor LILRA5 subfamily A member 5 A6NIT4 Chorionic somatomammotropin hormone 2 CSH2 isoform 2 A6NJ69 IgA-inducing protein homolog IGIP A6NKQ9 Choriogonadotropin subunit beta variant 1 CGB1 A6NMZ7 Collagen alpha-6(VI) chain COL6A6 A6NNS2 Dehydrogenase/reductase SDR family member 7C DHRS7C A6XGL2 Insulin A chain INS A8K0G1 Protein Wnt WNT7B A8K2U0 Alpha-2-macroglobulin-like protein 1 A2ML1 A8K7I4 Calcium-activated chloride channel regulator 1 CLCA1 A8MTL9 Serpin-like protein HMSD HMSD A8MV23 Serpin E3 SERPINE3 A8MZH6 Oocyte-secreted protein 1 homolog OOSP1 A8TX70 Collagen alpha-5(VI) chain COL6A5 B0ZBE8 Natriuretic peptide NPPA B1A4G9 Somatotropin GH1 B1A4H2 HCG1749481, isoform CRA_d CSH1 B1A4H9 Chorionic somatomammotropin hormone CSH2 B1AJZ6 Protein Wnt WNT4 B1AKI9 Isthmin-1 ISM1 B2RNN3 Complement C1q and tumor necrosis factor- C1QTNF9B related protein 9B B2RUY7 von Willebrand factor C domain-containing VWC2L protein 2-like B3GLJ2 Prostate and testis expressed protein 3 PATE3 B4DI03 SEC11-like 3 (S. cerevisiae), isoform CRA_a SEC11L3 B4DJF9 Protein Wnt WNT4 B4DUL4 SEC11-like 1 (S. cerevisiae), isoform CRA_d SEC11L1 B5MCC8 Protein Wnt WNT10B B8A595 Protein Wnt WNT7B B8A597 Protein Wnt WNT7B B8A598 Protein Wnt WNT7B B9A064 Immunoglobulin lambda-like polypeptide 5 IGLL5 C9J3H3 Protein Wnt WNT10B C9J8I8 Protein Wnt WNT5A C9JAF2 Insulin-like growth factor II Ala-25 Del IGF2 C9JCI2 Protein Wnt WNT10B C9JL84 HERV-H LTR-associating protein 1 HHLA1 C9JNR5 Insulin A chain INS C9JUI2 Protein Wnt WNT2 D6RF47 Protein Wnt WNT8A D6RF94 Protein Wnt WNT8A E2RYF7 Protein PBMUCL2 HCG22 E5RFR1 PENK(114-133) PENK E7EML9 Serine protease 44 PRSS44 E7EPC3 Protein Wnt WNT9B E7EVP0 Nociceptin PNOC E9PD02 Insulin-like growth factor I IGF1 E9PH60 Protein Wnt WNT16 E9PJL6 Protein Wnt WNT11 F5GYM2 Protein Wnt WNT5B F5H034 Protein Wnt WNT5B F5H364 Protein Wnt WNT5B F5H7Q6 Protein Wnt WNT5B F8WCM5 Protein INS-IGF2 INS-IGF2 F8WDR1 Protein Wnt WNT2 H0Y663 Protein Wnt WNT4 H0YK72 Signal peptidase complex catalytic subunit SEC11A SEC11A H0YK83 Signal peptidase complex catalytic subunit SEC11A SEC11A H0YM39 Chorionic somatomammotropin hormone CSH2 H0YMT7 Chorionic somatomammotropin hormone CSH1 H0YN17 Chorionic somatomammotropin hormone CSH2 H0YNA5 Signal peptidase complex catalytic subunit SEC11A SEC11A H0YNG3 Signal peptidase complex catalytic subunit SEC11A SEC11A H0YNX5 Signal peptidase complex catalytic subunit SEC11A SEC11A H7BZB8 Protein Wnt WNT10A H9KV56 Choriogonadotropin subunit beta variant 2 CGB2 I3L0L8 Protein Wnt WNT9B J3KNZ1 Choriogonadotropin subunit beta variant 1 CGB1 J3KP00 Choriogonadotropin subunit beta CGB7 J3QT02 Choriogonadotropin subunit beta variant 1 CGB1 O00175 C-C motif chemokine 24 CCL24 O00182 Galectin-9 LGALS9 O00187 Mannan-binding lectin serine protease 2 MASP2 O00230 Cortistatin CORT O00253 Agouti-related protein AGRP O00270 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid GPR31 receptor O00292 Left-right determination factor 2 LEFTY2 O00294 Tubby-related protein 1 TULP1 O00295 Tubby-related protein 2 TULP2 O00300 Tumor necrosis factor receptor superfamily TNFRSF11B member 11B O00339 Matrilin-2 MATN2 O00391 Sulfhydryl oxidase 1 QSOX1 O00468 Agrin AGRN O00515 Ladinin-1 LAD1 O00533 Processed neural cell adhesion molecule L1-like CHL1 protein O00584 Ribonuclease T2 RNASET2 O00585 C-C motif chemokine 21 CCL21 O00602 Ficolin-1 FCN1 O00622 Protein CYR61 CYR61 O00626 MDC(5-69) CCL22 O00634 Netrin-3 NTN3 O00744 Protein Wnt-10b WNT10B O00755 Protein Wnt-7a WNT7A O14498 Immunoglobulin superfamily containing leucine- ISLR rich repeat protein O14511 Pro-neuregulin-2, membrane-bound isoform NRG2 O14594 Neurocan core protein NCAN O14625 C-X-C motif chemokine 11 CXCL11 O14638 Ectonucleotide pyrophosphatase/phosphodiesterase ENPP3 family member 3 O14656 Torsin-1A TOR1A O14657 Torsin-1B TOR1B O14786 Neuropilin-1 NRP1 O14788 Tumor necrosis factor ligand superfamily member TNFSF11 11, membrane form O14791 Apolipoprotein L1 APOL1 O14793 Growth/differentiation factor 8 MSTN O14904 Protein Wnt-9a WNT9A O14905 Protein Wnt-9b WNT9B O14944 Proepiregulin EREG O14960 Leukocyte cell-derived chemotaxin-2 LECT2 O15018 Processed PDZ domain-containing protein 2 PDZD2 O15041 Semaphorin-3E SEMA3E O15072 A disintegrin and metalloproteinase with ADAMTS3 thrombospondin motifs 3 O15123 Angiopoietin-2 ANGPT2 O15130 Neuropeptide FF NPFF O15197 Ephrin type-B receptor 6 EPHB6 O15204 ADAM DEC1 ADAMDEC1 O15230 Laminin subunit alpha-5 LAMA5 O15232 Matrilin-3 MATN3 O15240 Neuroendocrine regulatory peptide-1 VGF O15263 Beta-defensin 4A DEFB4A O15335 Chondroadherin CHAD O15393 Transmembrane protease serine 2 catalytic chain TMPRSS2 O15444 C-C motif chemokine 25 CCL25 O15467 C-C motif chemokine 16 CCL16 O15496 Group 10 secretory phospholipase A2 PLA2G10 O15520 Fibroblast growth factor 10 FGF10 O15537 Retinoschisin RS1 O43157 Plexin-B1 PLXNB1 O43184 Disintegrin and metalloproteinase domain- ADAM12 containing protein 12 O43240 Kallikrein-10 KLK10 O43278 Kunitz-type protease inhibitor 1 SPINT1 O43320 Fibroblast growth factor 16 FGF16 O43323 Desert hedgehog protein C-product DHH O43405 Cochlin COCH O43508 Tumor necrosis factor ligand superfamily member TNFSF12 12, membrane form O43555 Progonadoliberin-2 GNRH2 O43557 Tumor necrosis factor ligand superfamily member TNFSF14 14, soluble form O43692 Peptidase inhibitor 15 PI15 O43699 Sialic acid-binding Ig-like lectin 6 SIGLEC6 O43820 Hyaluronidase-3 HYAL3 O43827 Angiopoietin-related protein 7 ANGPTL7 O43852 Calumenin CALU O43854 EGF-like repeat and discoidin I-like domain- EDIL3 containing protein 3 O43866 CD5 antigen-like CD5L O43897 Tolloid-like protein 1 TLL1 O43915 Vascular endothelial growth factor D FIGF O43927 C-X-C motif chemokine 13 CXCL13 O60218 Aldo-keto reductase family 1 member B10 AKR1B10 O60235 Transmembrane protease serine 11D TMPRSS11D O60258 Fibroblast growth factor 17 FGF17 O60259 Kallikrein-8 KLK8 O60383 Growth/differentiation factor 9 GDF9 O60469 Down syndrome cell adhesion molecule DSCAM O60542 Persephin PSPN O60565 Gremlin-1 GREM1 O60575 Serine protease inhibitor Kazal-type 4 SPINK4 O60676 Cystatin-8 CST8 O60687 Sushi repeat-containing protein SRPX2 SRPX2 O60844 Zymogen granule membrane protein 16 ZG16 O60882 Matrix metalloproteinase-20 MMP20 O60938 Keratocan KERA O75015 Low affinity immunoglobulin gamma Fc region FCGR3B receptor III-B O75077 Disintegrin and metalloproteinase domain- ADAM23 containing protein 23 O75093 Slit homolog 1 protein SLIT1 O75094 Slit homolog 3 protein SLIT3 O75095 Multiple epidermal growth factor-like domains MEGF6 protein 6 O75173 A disintegrin and metalloproteinase with ADAMTS4 thrombospondin motifs 4 O75200 Nuclear pore complex-interacting protein-like 1 NPIPL1 O75339 Cartilage intermediate layer protein 1 C1 CILP O75354 Ectonucleoside triphosphate diphosphohydrolase 6 ENTPD6 O75386 Tubby-related protein 3 TULP3 O75398 Deformed epidermal autoregulatory factor 1 DEAF1 homolog O75443 Alpha-tectorin TECTA O75445 Usherin USH2A O75462 Cytokine receptor-like factor 1 CRLF1 O75487 Glypican-4 GPC4 O75493 Carbonic anhydrase-related protein 11 CA11 O75594 Peptidoglycan recognition protein 1 PGLYRP1 O75596 C-type lectin domain family 3 member A CLEC3A O75610 Left-right determination factor 1 LEFTY1 O75629 Protein CREG1 CREG1 O75636 Ficolin-3 FCN3 O75711 Scrapie-responsive protein 1 SCRG1 O75715 Epididymal secretory glutathione peroxidase GPX5 O75718 Cartilage-associated protein CRTAP O75829 Chondrosurfactant protein LECT1 O75830 Serpin I2 SERPINI2 O75882 Attractin ATRN O75888 Tumor necrosis factor ligand superfamily TNFSF13 member 13 O75900 Matrix metalloproteinase-23 MMP23A O75951 Lysozyme-like protein 6 LYZL6 O75973 C1q-related factor C1QL1 O76038 Secretagogin SCGN O76061 Stanniocalcin-2 STC2 O76076 WNT1-inducible-signaling pathway protein 2 WISP2 O76093 Fibroblast growth factor 18 FGF18 O76096 Cystatin-F CST7 O94769 Extracellular matrix protein 2 ECM2 O94813 Slit homolog 2 protein C-product SLIT2 O94907 Dickkopf-related protein 1 DKK1 O94919 Endonuclease domain-containing 1 protein ENDOD1 O94964 N-terminal form SOGA1 O95025 Semaphorin-3D SEMA3D O95084 Serine protease 23 PRSS23 O95150 Tumor necrosis factor ligand superfamily TNFSF15 member 15 O95156 Neurexophilin-2 NXPH2 O95157 Neurexophilin-3 NXPH3 O95158 Neurexophilin-4 NXPH4 O95388 WNT1-inducible-signaling pathway protein 1 WISP1 O95389 WNT1-inducible-signaling pathway protein 3 WISP3 O95390 Growth/differentiation factor 11 GDF11 O95393 Bone morphogenetic protein 10 BMP10 O95399 Urotensin-2 UTS2 O95407 Tumor necrosis factor receptor superfamily TNFRSF6B member 6B O95428 Papilin PAPLN O95445 Apolipoprotein M APOM O95450 A disintegrin and metalloproteinase with ADAMTS2 thrombospondin motifs 2 O95460 Matrilin-4 MATN4 O95467 LHAL tetrapeptide GNAS O95631 Netrin-1 NTN1 O95633 Follistatin-related protein 3 FSTL3 O95711 Lymphocyte antigen 86 LY86 O95715 C-X-C motif chemokine 14 CXCL14 O95750 Fibroblast growth factor 19 FGF19 O95760 Interleukin-33 IL33 O95813 Cerberus CER1 O95841 Angiopoietin-related protein 1 ANGPTL1 O95897 Noelin-2 OLFM2 O95925 Eppin EPPIN O95965 Integrin beta-like protein 1 ITGBL1 O95967 EGF-containing fibulin-like extracellular matrix EFEMP2 protein 2 O95968 Secretoglobin family 1D member 1 SCGB1D1 O95969 Secretoglobin family 1D member 2 SCGB1D2 O95970 Leucine-rich glioma-inactivated protein 1 LGI1 O95972 Bone morphogenetic protein 15 BMP15 O95994 Anterior gradient protein 2 homolog AGR2 O95998 Interleukin-18-binding protein IL18BP O96009 Napsin-A NAPSA O96014 Protein Wnt-11 WNT11 P00450 Ceruloplasmin CP P00451 Factor VIIIa light chain F8 P00488 Coagulation factor XIII A chain F13A1 P00533 Epidermal growth factor receptor EGFR P00709 Alpha-lactalbumin LALBA P00734 Prothrombin F2 P00738 Haptoglobin beta chain HP P00739 Haptoglobin-related protein HPR P00740 Coagulation factor IXa heavy chain F9 P00742 Factor X heavy chain F10 P00746 Complement factor D CFD P00747 Plasmin light chain B PLG P00748 Coagulation factor XIIa light chain F12 P00749 Urokinase-type plasminogen activator long PLAU chain A P00750 Tissue-type plasminogen activator PLAT P00751 Complement factor B Ba fragment CFB P00797 Renin REN P00973 2′-5′-oligoadenylate synthase 1 OAS1 P00995 Pancreatic secretory trypsin inhibitor SPINK1 P01008 Antithrombin-III SERPINC1 P01009 Alpha-1-antitrypsin SERPINA1 P01011 Alpha-1-antichymotrypsin His-Pro-less SERPINA3 P01019 Angiotensin-1 AGT P01023 Alpha-2-macroglobulin A2M P01024 Acylation stimulating protein C3 P01031 Complement C5 beta chain C5 P01033 Metalloproteinase inhibitor 1 TIMP1 P01034 Cystatin-C CST3 P01036 Cystatin-S CST4 P01037 Cystatin-SN CST1 P01042 Kininogen-1 light chain KNG1 P01127 Platelet-derived growth factor subunit B PDGFB P01135 Transforming growth factor alpha TGFA P01137 Transforming growth factor beta-1 TGFB1 P01138 Beta-nerve growth factor NGF P01148 Gonadoliberin-1 GNRH1 P01160 Atrial natriuretic factor NPPA P01178 Oxytocin OXT P01185 Vasopressin-neurophysin 2-copeptin AVP P01189 Corticotropin POMC P01210 PENK(237-258) PENK P01213 Alpha-neoendorphin PDYN P01215 Glycoprotein hormones alpha chain CGA P01222 Thyrotropin subunit beta TSHB P01225 Follitropin subunit beta FSHB P01229 Lutropin subunit beta LHB P01233 Choriogonadotropin subunit beta CGB8 P01236 Prolactin PRL P01241 Somatotropin GH1 P01242 Growth hormone variant GH2 P01243 Chorionic somatomammotropin hormone CSH2 P01258 Katacalcin CALCA P01266 Thyroglobulin TG P01270 Parathyroid hormone PTH P01275 Glucagon GCG P01282 Intestinal peptide PHM-27 VIP P01286 Somatoliberin GHRH P01298 Pancreatic prohormone PPY P01303 C-flanking peptide of NPY NPY P01308 Insulin INS P01344 Insulin-like growth factor II IGF2 P01350 Big gastrin GAST P01374 Lymphotoxin-alpha LTA P01375 C-domain 1 TNF P01562 Interferon alpha-1/13 IFNA1 P01563 Interferon alpha-2 IFNA2 P01566 Interferon alpha-10 IFNA10 P01567 Interferon alpha-7 IFNA7 P01568 Interferon alpha-21 IFNA21 P01569 Interferon alpha-5 IFNA5 P01570 Interferon alpha-14 IFNA14 P01571 Interferon alpha-17 IFNA17 P01574 Interferon beta IFNB1 P01579 Interferon gamma IFNG P01583 Interleukin-1 alpha IL1A P01584 Interleukin-1 beta IL1B P01588 Erythropoietin EPO P01591 Immunoglobulin J chain IGJ P01732 T-cell surface glycoprotein CD8 alpha chain CD8A P01833 Polymeric immunoglobulin receptor PIGR P01857 Ig gamma-1 chain C region IGHG1 P01859 Ig gamma-2 chain C region IGHG2 P01860 Ig gamma-3 chain C region IGHG3 P01861 Ig gamma-4 chain C region IGHG4 P01871 Ig mu chain C region IGHM P01880 Ig delta chain C region IGHD P02452 Collagen alpha-1(I) chain COL1A1 P02458 Chondrocalcin COL2A1 P02461 Collagen alpha-1(III) chain COL3A1 P02462 Collagen alpha-1(IV) chain COL4A1 P02647 Apolipoprotein A-I APOA1 P02649 Apolipoprotein E APOE P02652 Apolipoprotein A-II APOA2 P02654 Apolipoprotein C-I APOC1 P02655 Apolipoprotein C-II APOC2 P02656 Apolipoprotein C-III APOC3 P02671 Fibrinogen alpha chain FGA P02675 Fibrinopeptide B FGB P02679 Fibrinogen gamma chain FGG P02741 C-reactive protein CRP P02743 Serum amyloid P-component(1-203) APCS P02745 Complement C1q subcomponent subunit A C1QA P02746 Complement C1q subcomponent subunit B C1QB P02747 Complement C1q subcomponent subunit C C1QC P02748 Complement component C9b C9 P02749 Beta-2-glycoprotein 1 APOH P02750 Leucine-rich alpha-2-glycoprotein LRG1 P02751 Ugl-Y2 FN1 P02753 Retinol-binding protein 4 RBP4 P02760 Trypstatin AMBP P02763 Alpha-1-acid glycoprotein 1 ORM1 P02765 Alpha-2-HS-glycoprotein chain A AHSG P02766 Transthyretin TTR P02768 Serum albumin ALB P02771 Alpha-fetoprotein AFP P02774 Vitamin D-binding protein GC P02775 Connective tissue-activating peptide III PPBP P02776 Platelet factor 4 PF4 P02778 CXCL10(1-73) CXCL10 P02786 Transferrin receptor protein 1 TFRC P02787 Serotransferrin TF P02788 Lactoferroxin-C LTF P02790 Hemopexin HPX P02808 Statherin STATH P02810 Salivary acidic proline-rich phosphoprotein 1/2 PRH2 P02812 Basic salivary proline-rich protein 2 PRB2 P02814 Peptide D1A SMR3B P02818 Osteocalcin BGLAP P03950 Angiogenin ANG P03951 Coagulation factor XIa heavy chain F11 P03952 Plasma kallikrein KLKB1 P03956 27 kDa interstitial collagenase MMP1 P03971 Muellerian-inhibiting factor AMH P03973 Antileukoproteinase SLPI P04003 C4b-binding protein alpha chain C4BPA P04004 Somatomedin-B VTN P04054 Phospholipase A2 PLA2G1B P04085 Platelet-derived growth factor subunit A PDGFA P04090 Relaxin A chain RLN2 P04114 Apolipoprotein B-100 APOB P04118 Colipase CLPS P04141 Granulocyte-macrophage colony-stimulating CSF2 factor P04155 Trefoil factor 1 TFF1 P04180 Phosphatidylcholine-sterol acyltransferase LCAT P04196 Histidine-rich glycoprotein HRG P04217 Alpha-1B-glycoprotein A1BG P04275 von Willebrand antigen 2 VWF P04278 Sex hormone-binding globulin SHBG P04279 Alpha-inhibin-31 SEMG1 P04280 Basic salivary proline-rich protein 1 PRB1 P04628 Proto-oncogene Wnt-1 WNT1 P04745 Alpha-amylase 1 AMY1A P04746 Pancreatic alpha-amylase AMY2A P04808 Prorelaxin H1 RLN1 P05000 Interferon omega-1 IFNW1 P05013 Interferon alpha-6 IFNA6 P05014 Interferon alpha-4 IFNA4 P05015 Interferon alpha-16 IFNA16 P05019 Insulin-like growth factor I IGF1 P05060 GAWK peptide CHGB P05090 Apolipoprotein D APOD P05109 Protein S100-A8 S100A8 P05111 Inhibin alpha chain INHA P05112 Interleukin-4 IL4 P05113 Interleukin-5 IL5 P05120 Plasminogen activator inhibitor 2 SERPINB2 P05121 Plasminogen activator inhibitor 1 SERPINE1 P05154 Plasma serine protease inhibitor SERPINA5 P05155 Plasma protease C1 inhibitor SERPING1 P05156 Complement factor I heavy chain CFI P05160 Coagulation factor XIII B chain F13B P05161 Ubiquitin-like protein ISG15 ISG15 P05230 Fibroblast growth factor 1 FGF1 P05231 Interleukin-6 IL6 P05305 Big endothelin-1 EDN1 P05408 C-terminal peptide SCG5 P05451 Lithostathine-1-alpha REG1A P05452 Tetranectin CLEC3B P05543 Thyroxine-binding globulin SERPINA7 P05814 Beta-casein CSN2 P05997 Collagen alpha-2(V) chain COL5A2 P06276 Cholinesterase BCHE P06307 Cholecystokinin-12 CCK P06396 Gelsolin GSN P06681 Complement C2 C2 P06702 Protein S100-A9 S100A9 P06727 Apolipoprotein A-IV APOA4 P06734 Low affinity immunoglobulin epsilon Fc receptor FCER2 soluble form P06744 Glucose-6-phosphate isomerase GPI P06850 Corticoliberin CRH P06858 Lipoprotein lipase LPL P06881 Calcitonin gene-related peptide 1 CALCA P07093 Glia-derived nexin SERPINE2 P07098 Gastric triacylglycerol lipase LIPF P07225 Vitamin K-dependent protein S PROS1 P07237 Protein disulfide-isomerase P4HB P07288 Prostate-specific antigen KLK3 P07306 Asialoglycoprotein receptor 1 ASGR1 P07355 Annexin A2 ANXA2 P07357 Complement component C8 alpha chain C8A P07358 Complement component C8 beta chain C8B P07360 Complement component C8 gamma chain C8G P07477 Alpha-trypsin chain 2 PRSS1 P07478 Trypsin-2 PRSS2 P07492 Neuromedin-C GRP P07498 Kappa-casein CSN3 P07585 Decorin DCN P07911 Uromodulin UMOD P07942 Laminin subunit beta-1 LAMB1 P07988 Pulmonary surfactant-associated protein B SFTPB P07998 Ribonuclease pancreatic RNASE1 P08118 Beta-microseminoprotein MSMB P08123 Collagen alpha-2(I) chain COL1A2 P08185 Corticosteroid-binding globulin SERPINA6 P08217 Chymotrypsin-like elastase family member 2A CELA2A P08218 Chymotrypsin-like elastase family member 2B CELA2B P08253 72 kDa type IV collagenase MMP2 P08254 Stromelysin-1 MMP3 P08294 Extracellular superoxide dismutase [Cu—Zn] SOD3 P08476 Inhibin beta A chain INHBA P08493 Matrix Gla protein MGP P08572 Collagen alpha-2(IV) chain COL4A2 P08581 Hepatocyte growth factor receptor MET P08603 Complement factor H CFH P08620 Fibroblast growth factor 4 FGF4 P08637 Low affinity immunoglobulin gamma Fc region FCGR3A receptor III-A P08697 Alpha-2-antiplasmin SERPINF2 P08700 Interleukin-3 IL3 P08709 Coagulation factor VII F7 P08833 Insulin-like growth factor-binding protein 1 IGFBP1 P08887 Interleukin-6 receptor subunit alpha IL6R P08949 Neuromedin-B-32 NMB P08F94 Fibrocystin PKHD1 P09038 Fibroblast growth factor 2 FGF2 P09228 Cystatin-SA CST2 P09237 Matrilysin MMP7 P09238 Stromelysin-2 MMP10 P09341 Growth-regulated alpha protein CXCL1 P09382 Galectin-1 LGALS1 P09466 Glycodelin PAEP P09486 SPARC SPARC P09529 Inhibin beta B chain INHBB P09544 Protein Wnt-2 WNT2 P09603 Processed macrophage colony-stimulating factor 1 CSF1 P09681 Gastric inhibitory polypeptide GIP P09683 Secretin SCT P09919 Granulocyte colony-stimulating factor CSF3 P0C091 FRAS1-related extracellular matrix protein 3 FREM3 P0C0L4 C4d-A C4A P0C0L5 Complement C4-B alpha chain C4B P0C0P6 Neuropeptide S NPS P0C7L1 Serine protease inhibitor Kazal-type 8 SPINK8 P0C862 Complement C1q and tumor necrosis factor- C1QTNF9 related protein 9A P0C8F1 Prostate and testis expressed protein 4 PATE4 P0CG01 Gastrokine-3 GKN3P P0CG36 Cryptic family protein 1B CFC1B P0CG37 Cryptic protein CFC1 P0CJ68 Humanin-like protein 1 MTRNR2L1 P0CJ69 Humanin-like protein 2 MTRNR2L2 P0CJ70 Humanin-like protein 3 MTRNR2L3 P0CJ71 Humanin-like protein 4 MTRNR2L4 P0CJ72 Humanin-like protein 5 MTRNR2L5 P0CJ73 Humanin-like protein 6 MTRNR2L6 P0CJ74 Humanin-like protein 7 MTRNR2L7 P0CJ75 Humanin-like protein 8 MTRNR2L8 P0CJ76 Humanin-like protein 9 MTRNR2L9 P0CJ77 Humanin-like protein 10 MTRNR2L10 P0DJD7 Pepsin A-4 PGA4 P0DJD8 Pepsin A-3 PGA3 P0DJD9 Pepsin A-5 PGA5 P0DJI8 Amyloid protein A SAA1 P0DJI9 Serum amyloid A-2 protein SAA2 P10082 Peptide YY(3-36) PYY P10092 Calcitonin gene-related peptide 2 CALCB P10124 Serglycin SRGN P10145 MDNCF-a IL8 P10147 MIP-1-alpha(4-69) CCL3 P10163 Peptide P-D PRB4 P10451 Osteopontin SPP1 P10599 Thioredoxin TXN P10600 Transforming growth factor beta-3 TGFB3 P10643 Complement component C7 C7 P10645 Vasostatin-2 CHGA P10646 Tissue factor pathway inhibitor TFPI P10720 Platelet factor 4 variant(4-74) PF4V1 P10745 Retinol-binding protein 3 RBP3 P10767 Fibroblast growth factor 6 FGF6 P10909 Clusterin alpha chain CLU P10912 Growth hormone receptor GHR P10915 Hyaluronan and proteoglycan link protein 1 HAPLN1 P10966 T-cell surface glycoprotein CD8 beta chain CD8B P10997 Islet amyloid polypeptide IAPP P11047 Laminin subunit gamma-1 LAMC1 P11150 Hepatic triacylglycerol lipase LIPC P11226 Mannose-binding protein C MBL2 P11464 Pregnancy-specific beta-1-glycoprotein 1 PSG1 P11465 Pregnancy-specific beta-1-glycoprotein 2 PSG2 P11487 Fibroblast growth factor 3 FGF3 P11597 Cholesteryl ester transfer protein CETP P11684 Uteroglobin SCGB1A1 P11686 Pulmonary surfactant-associated protein C SFTPC P12034 Fibroblast growth factor 5 FGF5 P12107 Collagen alpha-1(XI) chain COL11A1 P12109 Collagen alpha-1(VI) chain COL6A1 P12110 Collagen alpha-2(VI) chain COL6A2 P12111 Collagen alpha-3(VI) chain COL6A3 P12259 Coagulation factor V F5 P12272 PTHrP[1-36] PTHLH P12273 Prolactin-inducible protein PIP P12544 Granzyme A GZMA P12643 Bone morphogenetic protein 2 BMP2 P12644 Bone morphogenetic protein 4 BMP4 P12645 Bone morphogenetic protein 3 BMP3 P12724 Eosinophil cationic protein RNASE3 P12821 Angiotensin-converting enzyme, soluble form ACE P12838 Neutrophil defensin 4 DEFA4 P12872 Motilin MLN P13232 Interleukin-7 IL7 P13236 C-C motif chemokine 4 CCL4 P13284 Gamma-interferon-inducible lysosomal thiol IFI30 reductase P13500 C-C motif chemokine 2 CCL2 P13501 C-C motif chemokine 5 CCL5 P13521 Secretogranin-2 SCG2 P13591 Neural cell adhesion molecule 1 NCAM1 P13611 Versican core protein VCAN P13671 Complement component C6 C6 P13688 Carcinoembryonic antigen-related cell adhesion CEACAM1 molecule 1 P13725 Oncostatin-M OSM P13726 Tissue factor F3 P13727 Eosinophil granule major basic protein PRG2 P13942 Collagen alpha-2(XI) chain COL11A2 P13987 CD59 glycoprotein CD59 P14138 Endothelin-3 EDN3 P14174 Macrophage migration inhibitory factor MIF P14207 Folate receptor beta FOLR2 P14222 Perforin-1 PRF1 P14543 Nidogen-1 NID1 P14555 Phospholipase A2, membrane associated PLA2G2A P14625 Endoplasmin HSP90B1 P14735 Insulin-degrading enzyme IDE P14778 Interleukin-1 receptor type 1, soluble form IL1R1 P14780 82 kDa matrix metalloproteinase-9 MMP9 P15018 Leukemia inhibitory factor LIF P15085 Carboxypeptidase A1 CPA1 P15086 Carboxypeptidase B CPB1 P15151 Poliovirus receptor PVR P15169 Carboxypeptidase N catalytic chain CPN1 P15248 Interleukin-9 IL9 P15291 N-acetyllactosamine synthase B4GALT1 P15309 PAPf39 ACPP P15328 Folate receptor alpha FOLR1 P15374 Ubiquitin carboxyl-terminal hydrolase isozyme L3 UCHL3 P15502 Elastin ELN P15509 Granulocyte-macrophage colony-stimulating CSF2RA factor receptor subunit alpha P15515 Histatin-1 HTN1 P15516 His3-(31-51)-peptide HTN3 P15692 Vascular endothelial growth factor A VEGFA P15814 Immunoglobulin lambda-like polypeptide 1 IGLL1 P15907 Beta-galactoside alpha-2,6-sialyltransferase 1 ST6GAL1 P15941 Mucin-1 subunit beta MUC1 P16035 Metalloproteinase inhibitor 2 TIMP2 P16112 Aggrecan core protein 2 ACAN P16233 Pancreatic triacylglycerol lipase PNLIP P16442 Histo-blood group ABO system transferase ABO P16471 Prolactin receptor PRLR P16562 Cysteine-rich secretory protein 2 CRISP2 P16619 C-C motif chemokine 3-like 1 CCL3L1 P16860 BNP(3-29) NPPB P16870 Carboxypeptidase E CPE P16871 Interleukin-7 receptor subunit alpha IL7R P17213 Bactericidal permeability-increasing protein BPI P17538 Chymotrypsinogen B CTRB1 P17931 Galectin-3 LGALS3 P17936 Insulin-like growth factor-binding protein 3 IGFBP3 P17948 Vascular endothelial growth factor receptor 1 FLT1 P18065 Insulin-like growth factor-binding protein 2 IGFBP2 P18075 Bone morphogenetic protein 7 BMP7 P18428 Lipopolysaccharide-binding protein LBP P18509 PACAP-related peptide ADCYAP1 P18510 Interleukin-1 receptor antagonist protein IL1RN P18827 Syndecan-1 SDC1 P19021 Peptidylglycine alpha-hydroxylating PAM monooxygenase P19235 Erythropoietin receptor EPOR P19438 Tumor necrosis factor-binding protein 1 TNFRSF1A P19652 Alpha-1-acid glycoprotein 2 ORM2 P19801 Amiloride-sensitive amine oxidase [copper- ABP1 containing] P19823 Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 P19835 Bile salt-activated lipase CEL P19875 C-X-C motif chemokine 2 CXCL2 P19876 C-X-C motif chemokine 3 CXCL3 P19883 Follistatin FST P19957 Elafin PI3 P19961 Alpha-amylase 2B AMY2B P20061 Transcobalamin-1 TCN1 P20062 Transcobalamin-2 TCN2 P20142 Gastricsin PGC P20155 Serine protease inhibitor Kazal-type 2 SPINK2 P20231 Tryptase beta-2 TPSB2 P20333 Tumor necrosis factor receptor superfamily TNFRSF1B member 1B P20366 Substance P TAC1 P20382 Melanin-concentrating hormone PMCH P20396 Thyroliberin TRH P20742 Pregnancy zone protein PZP P20774 Mimecan OGN P20783 Neurotrophin-3 NTF3 P20800 Endothelin-2 EDN2 P20809 Interleukin-11 IL11 P20827 Ephrin-A1 EFNA1 P20849 Collagen alpha-1(IX) chain COL9A1 P20851 C4b-binding protein beta chain C4BPB P20908 Collagen alpha-1(V) chain COL5A1 P21128 Poly(U)-specific endoribonuclease ENDOU P21246 Pleiotrophin PTN P21583 Kit ligand KITLG P21741 Midkine MDK P21754 Zona pellucida sperm-binding protein 3 ZP3 P21781 Fibroblast growth factor 7 FGF7 P21802 Fibroblast growth factor receptor 2 FGFR2 P21810 Biglycan BGN P21815 Bone sialoprotein 2 IBSP P21860 Receptor tyrosine-protein kinase erbB-3 ERBB3 P21941 Cartilage matrix protein MATN1 P22003 Bone morphogenetic protein 5 BMP5 P22004 Bone morphogenetic protein 6 BMP6 P22079 Lactoperoxidase LPO P22105 Tenascin-X TNXB P22301 Interleukin-10 IL10 P22303 Acetylcholinesterase ACHE P22352 Glutathione peroxidase 3 GPX3 P22362 C-C motif chemokine 1 CCL1 P22455 Fibroblast growth factor receptor 4 FGFR4 P22466 Galanin message-associated peptide GAL P22692 Insulin-like growth factor-binding protein 4 IGFBP4 P22749 Granulysin GNLY P22792 Carboxypeptidase N subunit 2 CPN2 P22891 Vitamin K-dependent protein Z PROZ P22894 Neutrophil collagenase MMP8 P23142 Fibulin-1 FBLN1 P23280 Carbonic anhydrase 6 CA6 P23352 Anosmin-1 KAL1 P23435 Cerebellin-1 CBLN1 P23560 Brain-derived neurotrophic factor BDNF P23582 C-type natriuretic peptide NPPC P23946 Chymase CMA1 P24043 Laminin subunit alpha-2 LAMA2 P24071 Immunoglobulin alpha Fc receptor FCAR P24347 Stromelysin-3 MMP11 P24387 Corticotropin-releasing factor-binding protein CRHBP P24592 Insulin-like growth factor-binding protein 6 IGFBP6 P24593 Insulin-like growth factor-binding protein 5 IGFBP5 P24821 Tenascin TNC P24855 Deoxyribonuclease-1 DNASE1 P25067 Collagen alpha-2(VIII) chain COL8A2 P25311 Zinc-alpha-2-glycoprotein AZGP1 P25391 Laminin subunit alpha-1 LAMA1 P25445 Tumor necrosis factor receptor superfamily FAS member 6 P25940 Collagen alpha-3(V) chain COL5A3 P25942 Tumor necrosis factor receptor superfamily CD40 member 5 P26022 Pentraxin-related protein PTX3 PTX3 P26927 Hepatocyte growth factor-like protein beta chain MST1 P27169 Serum paraoxonase/arylesterase 1 PON1 P27352 Gastric intrinsic factor GIF P27487 Dipeptidyl peptidase 4 membrane form DPP4 P27539 Embryonic growth/differentiation factor 1 GDF1 P27658 Vastatin COL8A1 P27797 Calreticulin CALR P27918 Properdin CFP P28039 Acyloxyacyl hydrolase AOAH P28300 Protein-lysine 6-oxidase LOX P28325 Cystatin-D CST5 P28799 Granulin-1 GRN P29122 Proprotein convertase subtilisin/kexin type 6 PCSK6 P29279 Connective tissue growth factor CTGF P29320 Ephrin type-A receptor 3 EPHA3 P29400 Collagen alpha-5(IV) chain COL4A5 P29459 Interleukin-12 subunit alpha IL12A P29460 Interleukin-12 subunit beta IL12B P29508 Serpin B3 SERPINB3 P29622 Kallistatin SERPINA4 P29965 CD40 ligand, soluble form CD40LG P30990 Neurotensin/neuromedin N NTS P31025 Lipocalin-1 LCN1 P31151 Protein S100-A7 S100A7 P31371 Fibroblast growth factor 9 FGF9 P31431 Syndecan-4 SDC4 P31947 14-3-3 protein sigma SFN P32455 Interferon-induced guanylate-binding protein 1 GBP1 P32881 Interferon alpha-8 IFNA8 P34096 Ribonuclease 4 RNASE4 P34130 Neurotrophin-4 NTF4 P34820 Bone morphogenetic protein 8B BMP8B P35030 Trypsin-3 PRSS3 P35052 Secreted glypican-1 GPC1 P35070 Betacellulin BTC P35225 Interleukin-13 IL13 P35247 Pulmonary surfactant-associated protein D SFTPD P35318 ADM ADM P35542 Serum amyloid A-4 protein SAA4 P35555 Fibrillin-1 FBN1 P35556 Fibrillin-2 FBN2 P35625 Metalloproteinase inhibitor 3 TIMP3 P35858 Insulin-like growth factor-binding protein complex IGFALS acid labile subunit P35916 Vascular endothelial growth factor receptor 3 FLT4 P35968 Vascular endothelial growth factor receptor 2 KDR P36222 Chitinase-3-like protein 1 CHI3L1 P36952 Serpin B5 SERPINB5 P36955 Pigment epithelium-derived factor SERPINF1 P36980 Complement factor H-related protein 2 CFHR2 P39059 Collagen alpha-1(XV) chain COL15A1 P39060 Collagen alpha-1(XVIII) chain COL18A1 P39877 Calcium-dependent phospholipase A2 PLA2G5 P39900 Macrophage metalloelastase MMP12 P39905 Glial cell line-derived neurotrophic factor GDNF P40225 Thrombopoietin THPO P40967 M-alpha PMEL P41159 Leptin LEP P41221 Protein Wnt-5a WNT5A P41222 Prostaglandin-H2 D-isomerase PTGDS P41271 Neuroblastoma suppressor of tumorigenicity 1 NBL1 P41439 Folate receptor gamma FOLR3 P42127 Agouti-signaling protein ASIP P42702 Leukemia inhibitory factor receptor LIFR P42830 ENA-78(9-78) CXCL5 P43026 Growth/differentiation factor 5 GDF5 P43251 Biotinidase BTD P43652 Afamin AFM P45452 Collagenase 3 MMP13 P47710 Casoxin-D CSN1S1 P47929 Galectin-7 LGALS7B P47972 Neuronal pentraxin-2 NPTX2 P47989 Xanthine oxidase XDH P47992 Lymphotactin XCL1 P48023 Tumor necrosis factor ligand superfamily FASLG member 6, membrane form P48052 Carboxypeptidase A2 CPA2 P48061 Stromal cell-derived factor 1 CXCL12 P48304 Lithostathine-1-beta REG1B P48307 Tissue factor pathway inhibitor 2 TFPI2 P48357 Leptin receptor LEPR P48594 Serpin B4 SERPINB4 P48645 Neuromedin-U-25 NMU P48740 Mannan-binding lectin serine protease 1 MASP1 P48745 Protein NOV homolog NOV P48960 CD97 antigen subunit beta CD97 P49223 Kunitz-type protease inhibitor 3 SPINT3 P49747 Cartilage oligomeric matrix protein COMP P49763 Placenta growth factor PGF P49765 Vascular endothelial growth factor B VEGFB P49767 Vascular endothelial growth factor C VEGFC P49771 Fms-related tyrosine kinase 3 ligand FLT3LG P49862 Kallikrein-7 KLK7 P49863 Granzyme K GZMK P49908 Selenoprotein P SEPP1 P49913 Antibacterial protein FALL-39 CAMP P50607 Tubby protein homolog TUB P51124 Granzyme M GZMM P51512 Matrix metalloproteinase-16 MMP16 P51654 Glypican-3 GPC3 P51671 Eotaxin CCL11 P51884 Lumican LUM P51888 Prolargin PRELP P52798 Ephrin-A4 EFNA4 P52823 Stanniocalcin-1 STC1 P53420 Collagen alpha-4(IV) chain COL4A4 P53621 Coatomer subunit alpha COPA P54108 Cysteine-rich secretory protein 3 CRISP3 P54315 Pancreatic lipase-related protein 1 PNLIPRP1 P54317 Pancreatic lipase-related protein 2 PNLIPRP2 P54793 Arylsulfatase F ARSF P55000 Secreted Ly-6/uPAR-related protein 1 SLURP1 P55001 Microfibrillar-associated protein 2 MFAP2 P55056 Apolipoprotein C-IV APOC4 P55058 Phospholipid transfer protein PLTP P55075 Fibroblast growth factor 8 FGF8 P55081 Microfibrillar-associated protein 1 MFAP1 P55083 Microfibril-associated glycoprotein 4 MFAP4 P55107 Bone morphogenetic protein 3B GDF10 P55145 Mesencephalic astrocyte-derived neurotrophic MANF factor P55259 Pancreatic secretory granule membrane major GP2 glycoprotein GP2 P55268 Laminin subunit beta-2 LAMB2 P55773 CCL23(30-99) CCL23 P55774 C-C motif chemokine 18 CCL18 P55789 FAD-linked sulfhydryl oxidase ALR GFER P56703 Proto-oncogene Wnt-3 WNT3 P56704 Protein Wnt-3a WNT3A P56705 Protein Wnt-4 WNT4 P56706 Protein Wnt-7b WNT7B P56730 Neurotrypsin PRSS12 P56851 Epididymal secretory protein E3-beta EDDM3B P56975 Neuregulin-3 NRG3 P58062 Serine protease inhibitor Kazal-type 7 SPINK7 P58215 Lysyl oxidase homolog 3 LOXL3 P58294 Prokineticin-1 PROK1 P58335 Anthrax toxin receptor 2 ANTXR2 P58397 A disintegrin and metalloproteinase with ADAMTS12 thrombospondin motifs 12 P58417 Neurexophilin-1 NXPH1 P58499 Protein FAM3B FAM3B P59510 A disintegrin and metalloproteinase with ADAMTS20 thrombospondin motifs 20 P59665 Neutrophil defensin 1 DEFA1B P59666 Neutrophil defensin 3 DEFA3 P59796 Glutathione peroxidase 6 GPX6 P59826 BPI fold-containing family B member 3 BPIFB3 P59827 BPI fold-containing family B member 4 BPIFB4 P59861 Beta-defensin 131 DEFB131 P60022 Beta-defensin 1 DEFB1 P60153 Inactive ribonuclease-like protein 9 RNASE9 P60827 Complement C1q tumor necrosis factor-related C1QTNF8 protein 8 P60852 Zona pellucida sperm-binding protein 1 ZP1 P60985 Keratinocyte differentiation-associated protein KRTDAP P61109 Kidney androgen-regulated protein KAP P61278 Somatostatin-14 SST P61366 Osteocrin OSTN P61626 Lysozyme C LYZ P61769 Beta-2-microglobulin B2M P61812 Transforming growth factor beta-2 TGFB2 P61916 Epididymal secretory protein E1 NPC2 P62502 Epididymal-specific lipocalin-6 LCN6 P62937 Peptidyl-prolyl cis-trans isomerase A PPIA P67809 Nuclease-sensitive element-binding protein 1 YBX1 P67812 Signal peptidase complex catalytic subunit SEC11A SEC11A P78310 Coxsackievirus and adenovirus receptor CXADR P78333 Secreted glypican-5 GPC5 P78380 Oxidized low-density lipoprotein receptor 1 OLR1 P78423 Processed fractalkine CX3CL1 P78509 Reelin RELN P78556 CCL20(2-70) CCL20 P80075 MCP-2(6-76) CCL8 P80098 C-C motif chemokine 7 CCL7 P80108 Phosphatidylinositol-glycan-specific GPLD1 phospholipase D P80162 C-X-C motif chemokine 6 CXCL6 P80188 Neutrophil gelatinase-associated lipocalin LCN2 P80303 Nucleobindin-2 NUCB2 P80511 Calcitermin S100A12 P81172 Hepcidin-25 HAMP P81277 Prolactin-releasing peptide PRLH P81534 Beta-defensin 103 DEFB103A P81605 Dermcidin DCD P82279 Protein crumbs homolog 1 CRB1 P82987 ADAMTS-like protein 3 ADAMTSL3 P83105 Serine protease HTRA4 HTRA4 P83110 Serine protease HTRA3 HTRA3 P83859 Orexigenic neuropeptide QRFP QRFP P98088 Mucin-5AC MUC5AC P98095 Fibulin-2 FBLN2 P98160 Basement membrane-specific heparan sulfate HSPG2 proteoglycan core protein P98173 Protein FAM3A FAM3A Q00604 Norrin NDP Q00796 Sorbitol dehydrogenase SORD Q00887 Pregnancy-specific beta-1-glycoprotein 9 PSG9 Q00888 Pregnancy-specific beta-1-glycoprotein 4 PSG4 Q00889 Pregnancy-specific beta-1-glycoprotein 6 PSG6 Q01523 HD5(56-94) DEFA5 Q01524 Defensin-6 DEFA6 Q01955 Collagen alpha-3(IV) chain COL4A3 Q02297 Pro-neuregulin-1, membrane-bound isoform NRG1 Q02325 Plasminogen-like protein B PLGLB1 Q02383 Semenogelin-2 SEMG2 Q02388 Collagen alpha-1(VII) chain COL7A1 Q02505 Mucin-3A MUC3A Q02509 Otoconin-90 OC90 Q02747 Guanylin GUCA2A Q02763 Angiopoietin-1 receptor TEK Q02817 Mucin-2 MUC2 Q02985 Complement factor H-related protein 3 CFHR3 Q03167 Transforming growth factor beta receptor type 3 TGFBR3 Q03403 Trefoil factor 2 TFF2 Q03405 Urokinase plasminogen activator surface receptor PLAUR Q03591 Complement factor H-related protein 1 CFHR1 Q03692 Collagen alpha-1(X) chain COL10A1 Q04118 Basic salivary proline-rich protein 3 PRB3 Q04756 Hepatocyte growth factor activator short chain HGFAC Q04900 Sialomucin core protein 24 CD164 Q05315 Eosinophil lysophospholipase CLC Q05707 Collagen alpha-1(XIV) chain COL14A1 Q05996 Processed zona pellucida sperm-binding protein 2 ZP2 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 Q06141 Regenerating islet-derived protein 3-alpha REG3A Q06828 Fibromodulin FMOD Q07092 Collagen alpha-1(XVI) chain COL16A1 Q07325 C-X-C motif chemokine 9 CXCL9 Q07507 Dermatopontin DPT Q075Z2 Binder of sperm protein homolog 1 BSPH1 Q07654 Trefoil factor 3 TFF3 Q07699 Sodium channel subunit beta-1 SCN1B Q08345 Epithelial discoidin domain-containing receptor 1 DDR1 Q08380 Galectin-3-binding protein LGALS3BP Q08397 Lysyl oxidase homolog 1 LOXL1 Q08431 Lactadherin MFGE8 Q08629 Testican-1 SPOCK1 Q08648 Sperm-associated antigen 11B SPAG11B Q08830 Fibrinogen-like protein 1 FGL1 Q10471 Polypeptide N-acetylgalactosaminyltransferase 2 GALNT2 Q10472 Polypeptide N-acetylgalactosaminyltransferase 1 GALNT1 Q11201 CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL1 alpha-2,3-sialyltransferase 1 Q11203 CMP-N-acetylneuraminate-beta-1,4-galactoside ST3GAL3 alpha-2,3-sialyltransferase Q11206 CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL4 alpha-2,3-sialyltransferase 4 Q12794 Hyaluronidase-1 HYAL1 Q12805 EGF-containing fibulin-like extracellular matrix EFEMP1 protein 1 Q12836 Zona pellucida sperm-binding protein 4 ZP4 Q12841 Follistatin-related protein 1 FSTL1 Q12904 Aminoacyl tRNA synthase complex-interacting AIMP1 multifunctional protein 1 Q13018 Soluble secretory phospholipase A2 receptor PLA2R1 Q13072 B melanoma antigen 1 BAGE Q13093 Platelet-activating factor acetylhydrolase PLA2G7 Q13103 Secreted phosphoprotein 24 SPP2 Q13162 Peroxiredoxin-4 PRDX4 Q13201 Platelet glycoprotein Ia* MMRN1 Q13214 Semaphorin-3B SEMA3B Q13219 Pappalysin-1 PAPPA Q13231 Chitotriosidase-1 CHIT1 Q13253 Noggin NOG Q13261 Interleukin-15 receptor subunit alpha IL15RA Q13275 Semaphorin-3F SEMA3F Q13291 Signaling lymphocytic activation molecule SLAMF1 Q13316 Dentin matrix acidic phosphoprotein 1 DMP1 Q13361 Microfibrillar-associated protein 5 MFAP5 Q13410 Butyrophilin subfamily 1 member A1 BTN1A1 Q13421 Mesothelin, cleaved form MSLN Q13429 Insulin-like growth factor I IGF-I Q13443 Disintegrin and metalloproteinase domain- ADAM9 containing protein 9 Q13519 Neuropeptide 1 PNOC Q13751 Laminin subunit beta-3 LAMB3 Q13753 Laminin subunit gamma-2 LAMC2 Q13790 Apolipoprotein F APOF Q13822 Ectonucleotide pyrophosphatase/phosphodiesterase ENPP2 family member 2 Q14031 Collagen alpha-6(IV) chain COL4A6 Q14050 Collagen alpha-3(IX) chain COL9A3 Q14055 Collagen alpha-2(IX) chain COL9A2 Q14112 Nidogen-2 NID2 Q14114 Low-density lipoprotein receptor-related protein 8 LRP8 Q14118 Dystroglycan DAG1 Q14314 Fibroleukin FGL2 Q14393 Growth arrest-specific protein 6 GAS6 Q14406 Chorionic somatomammotropin hormone-like 1 CSHL1 Q14507 Epididymal secretory protein E3-alpha EDDM3A Q14508 WAP four-disulfide core domain protein 2 WFDC2 Q14512 Fibroblast growth factor-binding protein 1 FGFBP1 Q14515 SPARC-like protein 1 SPARCL1 Q14520 Hyaluronan-binding protein 2 27 kDa light chain HABP2 Q14563 Semaphorin-3A SEMA3A Q14623 Indian hedgehog protein IHH Q14624 Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 Q14667 UPF0378 protein KIAA0100 KIAA0100 Q14703 Membrane-bound transcription factor site-1 MBTPS1 protease Q14766 Latent-transforming growth factor beta-binding LTBP1 protein 1 Q14767 Latent-transforming growth factor beta-binding LTBP2 protein 2 Q14773 Intercellular adhesion molecule 4 ICAM4 Q14993 Collagen alpha-1(XIX) chain COL19A1 Q14CN2 Calcium-activated chloride channel regulator 4, CLCA4 110 kDa form Q15046 Lysine--tRNA ligase KARS Q15063 Periostin POSTN Q15109 Advanced glycosylation end product-specific AGER receptor Q15113 Procollagen C-endopeptidase enhancer 1 PCOLCE Q15166 Serum paraoxonase/lactonase 3 PON3 Q15195 Plasminogen-like protein A PLGLA Q15198 Platelet-derived growth factor receptor-like protein PDGFRL Q15223 Poliovirus receptor-related protein 1 PVRL1 Q15238 Pregnancy-specific beta-1-glycoprotein 5 PSG5 Q15363 Transmembrane emp24 domain-containing protein 2 TMED2 Q15375 Ephrin type-A receptor 7 EPHA7 Q15389 Angiopoietin-1 ANGPT1 Q15465 Sonic hedgehog protein SHH Q15485 Ficolin-2 FCN2 Q15517 Corneodesmosin CDSN Q15582 Transforming growth factor-beta-induced protein ig-h3 TGFBI Q15661 Tryptase alpha/beta-1 TPSAB1 Q15726 Metastin KISS1 Q15782 Chitinase-3-like protein 2 CHI3L2 Q15828 Cystatin-M CST6 Q15846 Clusterin-like protein 1 CLUL1 Q15848 Adiponectin ADIPOQ Q16206 Protein disulfide-thiol oxidoreductase ENOX2 Q16270 Insulin-like growth factor-binding protein 7 IGFBP7 Q16363 Laminin subunit alpha-4 LAMA4 Q16378 Proline-rich protein 4 PRR4 Q16557 Pregnancy-specific beta-1-glycoprotein 3 PSG3 Q16568 CART(42-89) CARTPT Q16610 Extracellular matrix protein 1 ECM1 Q16619 Cardiotrophin-1 CTF1 Q16623 Syntaxin-1A STX1A Q16627 HCC-1(9-74) CCL14 Q16651 Prostasin light chain PRSS8 Q16661 Guanylate cyclase C-activating peptide 2 GUCA2B Q16663 CCL15(29-92) CCL15 Q16674 Melanoma-derived growth regulatory protein MIA Q16769 Glutaminyl-peptide cyclotransferase QPCT Q16787 Laminin subunit alpha-3 LAMA3 Q16842 CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL2 alpha-2,3-sialyltransferase 2 Q17RR3 Pancreatic lipase-related protein 3 PNLIPRP3 Q17RW2 Collagen alpha-1(XXIV) chain COL24A1 Q17RY6 Lymphocyte antigen 6K LY6K Q1L6U9 Prostate-associated microseminoprotein MSMP Q1W4C9 Serine protease inhibitor Kazal-type 13 SPINK13 Q1ZYL8 Izumo sperm-egg fusion protein 4 IZUMO4 Q29960 HLA class I histocompatibility antigen, Cw-16 HLA-C alpha chain Q2I0M5 R-spondin-4 RSPO4 Q2L4Q9 Serine protease 53 PRSS53 Q2MKA7 R-spondin-1 RSPO1 Q2MV58 Tectonic-1 TCTN1 Q2TAL6 Brorin VWC2 Q2UY09 Collagen alpha-1(XXVIII) chain COL28A1 Q2VPA4 Complement component receptor 1-like protein CR1L Q2WEN9 Carcinoembryonic antigen-related cell adhesion CEACAM16 molecule 16 Q30KP8 Beta-defensin 136 DEFB136 Q30KP9 Beta-defensin 135 DEFB135 Q30KQ1 Beta-defensin 133 DEFB133 Q30KQ2 Beta-defensin 130 DEFB130 Q30KQ4 Beta-defensin 116 DEFB116 Q30KQ5 Beta-defensin 115 DEFB115 Q30KQ6 Beta-defensin 114 DEFB114 Q30KQ7 Beta-defensin 113 DEFB113 Q30KQ8 Beta-defensin 112 DEFB112 Q30KQ9 Beta-defensin 110 DEFB110 Q30KR1 Beta-defensin 109 DEFB109P1 Q32P28 Prolyl 3-hydroxylase 1 LEPRE1 Q3B7J2 Glucose-fructose oxidoreductase domain- GFOD2 containing protein 2 Q3SY79 Protein Wnt WNT3A Q3T906 N-acetylglucosamine-1-phosphotransferase GNPTAB subunits alpha/beta Q495T6 Membrane metallo-endopeptidase-like 1 MMEL1 Q49AH0 Cerebral dopamine neurotrophic factor CDNF Q4G0G5 Secretoglobin family 2B member 2 SCGB2B2 Q4G0M1 Protein FAM132B FAM132B Q4LDE5 Sushi, von Willebrand factor type A, EGF and SVEP1 pentraxin domain-containing protein 1 Q4QY38 Beta-defensin 134 DEFB134 Q4VAJ4 Protein Wnt WNT10B Q4W5P6 Protein TMEM155 TMEM155 Q4ZHG4 Fibronectin type III domain-containing protein 1 FNDC1 Q53H76 Phospholipase A1 member A PLA1A Q53RD9 Fibulin-7 FBLN7 Q53S33 BolA-like protein 3 BOLA3 Q5BLP8 Neuropeptide-like protein C4orf48 C4orf48 Q5DT21 Serine protease inhibitor Kazal-type 9 SPINK9 Q5EBL8 PDZ domain-containing protein 11 PDZD11 Q5FYB0 Arylsulfatase J ARSJ Q5FYB1 Arylsulfatase I ARSI Q5GAN3 Ribonuclease-like protein 13 RNASE13 Q5GAN4 Ribonuclease-like protein 12 RNASE12 Q5GAN6 Ribonuclease-like protein 10 RNASE10 Q5GFL6 von Willebrand factor A domain-containing VWA2 protein 2 Q5H8A3 Neuromedin-S NMS Q5H8C1 FRAS1-related extracellular matrix protein 1 FREM1 Q5IJ48 Protein crumbs homolog 2 CRB2 Q5J5C9 Beta-defensin 121 DEFB121 Q5JS37 NHL repeat-containing protein 3 NHLRC3 Q5JTB6 Placenta-specific protein 9 PLAC9 Q5JU69 Torsin-2A TOR2A Q5JXM2 Methyltransferase-like protein 24 METTL24 Q5JZY3 Ephrin type-A receptor 10 EPHA10 Q5K4E3 Polyserase-2 PRSS36 Q5SRR4 Lymphocyte antigen 6 complex locus protein G5c LY6G5C Q5T1H1 Protein eyes shut homolog EYS Q5T4F7 Secreted frizzled-related protein 5 SFRP5 Q5T4W7 Artemin ARTN Q5T7M4 Protein FAM132A FAM132A Q5TEH8 Protein Wnt WNT2B Q5TIE3 von Willebrand factor A domain-containing VWA5B1 protein 5B1 Q5UCC4 ER membrane protein complex subunit 10 EMC10 Q5VST6 Abhydrolase domain-containing protein FAM108B1 FAM108B1 Q5VTL7 Fibronectin type III domain-containing protein 7 FNDC7 Q5VUM1 UPF0369 protein C6orf57 C6orf57 Q5VV43 Dyslexia-associated protein KIAA0319 KIAA0319 Q5VWW1 Complement C1q-like protein 3 C1QL3 Q5VXI9 Lipase member N LIPN Q5VXJ0 Lipase member K LIPK Q5VXM1 CUB domain-containing protein 2 CDCP2 Q5VYX0 Renalase RNLS Q5VYY2 Lipase member M LIPM Q5W186 Cystatin-9 CST9 Q5W5W9 Regulated endocrine-specific protein 18 RESP18 Q5XG92 Carboxylesterase 4A CES4A Q63HQ2 Pikachurin EGFLAM Q641Q3 Meteorin-like protein METRNL Q66K79 Carboxypeptidase Z CPZ Q685J3 Mucin-17 MUC17 Q68BL7 Olfactomedin-like protein 2A OLFML2A Q68BL8 Olfactomedin-like protein 2B OLFML2B Q68DV7 E3 ubiquitin-protein ligase RNF43 RNF43 Q6B9Z1 Insulin growth factor-like family member 4 IGFL4 Q6BAA4 Fc receptor-like B FCRLB Q6E0U4 Dermokine DMKN Q6EMK4 Vasorin VASN Q6FHJ7 Secreted frizzled-related protein 4 SFRP4 Q6GPI1 Chymotrypsin B2 chain B CTRB2 Q6GTS8 Probable carboxypeptidase PM20D1 PM20D1 Q6H9L7 Isthmin-2 ISM2 Q6IE36 Ovostatin homolog 2 OVOS2 Q6IE37 Ovostatin homolog 1 OVOS1 Q6IE38 Serine protease inhibitor Kazal-type 14 SPINK14 Q6ISS4 Leukocyte-associated immunoglobulin-like LAIR2 receptor 2 Q6JVE5 Epididymal-specific lipocalin-12 LCN12 Q6JVE6 Epididymal-specific lipocalin-10 LCN10 Q6JVE9 Epididymal-specific lipocalin-8 LCN8 Q6KF10 Growth/differentiation factor 6 GDF6 Q6MZW2 Follistatin-related protein 4 FSTL4 Q6NSX1 Coiled-coil domain-containing protein 70 CCDC70 Q6NT32 Carboxylesterase 5A CES5A Q6NT52 Choriogonadotropin subunit beta variant 2 CGB2 Q6NUI6 Chondroadherin-like protein CHADL Q6NUJ1 Saposin A-like PSAPL1 Q6P093 Arylacetamide deacetylase-like 2 AADACL2 Q6P4A8 Phospholipase B-like 1 PLBD1 Q6P5S2 UPF0762 protein C6orf58 C6orf58 Q6P988 Protein notum homolog NOTUM Q6PCB0 von Willebrand factor A domain-containing VWA1 protein 1 Q6PDA7 Sperm-associated antigen 11A SPAG11A Q6PEW0 Inactive serine protease 54 PRSS54 Q6PEZ8 Podocan-like protein 1 PODNL1 Q6PKH6 Dehydrogenase/reductase SDR family member 4- DHRS4L2 like 2 Q6Q788 Apolipoprotein A-V APOA5 Q6SPF0 Atherin SAMD1 Q6UDR6 Kunitz-type protease inhibitor 4 SPINT4 Q6URK8 Testis, prostate and placenta-expressed protein TEPP Q6UW01 Cerebellin-3 CBLN3 Q6UW10 Surfactant-associated protein 2 SFTA2 Q6UW15 Regenerating islet-derived protein 3-gamma REG3G Q6UW32 Insulin growth factor-like family member 1 IGFL1 Q6UW78 UPF0723 protein C11orf83 C11orf83 Q6UW88 Epigen EPGN Q6UWE3 Colipase-like protein 2 CLPSL2 Q6UWF7 NXPE family member 4 NXPE4 Q6UWF9 Protein FAM180A FAM180A Q6UWM5 GLIPR1-like protein 1 GLIPR1L1 Q6UWN8 Serine protease inhibitor Kazal-type 6 SPINK6 Q6UWP2 Dehydrogenase/reductase SDR family member 11 DHRS11 Q6UWP8 Suprabasin SBSN Q6UWQ5 Lysozyme-like protein 1 LYZL1 Q6UWQ7 Insulin growth factor-like family member 2 IGFL2 Q6UWR7 Ectonucleotide pyrophosphatase/phosphodiesterase ENPP6 family member 6 soluble form Q6UWT2 Adropin ENHO Q6UWU2 Beta-galactosidase-1-like protein GLB1L Q6UWW0 Lipocalin-15 LCN15 Q6UWX4 HHIP-like protein 2 HHIPL2 Q6UWY0 Arylsulfatase K ARSK Q6UWY2 Serine protease 57 PRSS57 Q6UWY5 Olfactomedin-like protein 1 OLFML1 Q6UX06 Olfactomedin-4 OLFM4 Q6UX07 Dehydrogenase/reductase SDR family member 13 DHRS13 Q6UX39 Amelotin AMTN Q6UX46 Protein FAM150B FAM150B Q6UX73 UPF0764 protein C16orf89 C16orf89 Q6UXB0 Protein FAM131A FAM131A Q6UXB1 Insulin growth factor-like family member 3 IGFL3 Q6UXB2 VEGF co-regulated chemokine 1 CXCL17 Q6UXF7 C-type lectin domain family 18 member B CLEC18B Q6UXH0 Hepatocellular carcinoma-associated protein TD26 C19orf80 Q6UXH1 Cysteine-rich with EGF-like domain protein 2 CRELD2 Q6UXH8 Collagen and calcium-binding EGF domain- CCBE1 containing protein 1 Q6UXH9 Inactive serine protease PAMR1 PAMR1 Q6UXI7 Vitrin VIT Q6UXI9 Nephronectin NPNT Q6UXN2 Trem-like transcript 4 protein TREML4 Q6UXS0 C-type lectin domain family 19 member A CLEC19A Q6UXT8 Protein FAM150A FAM150A Q6UXT9 Abhydrolase domain-containing protein 15 ABHD15 Q6UXV4 Apolipoprotein O-like APOOL Q6UXX5 Inter-alpha-trypsin inhibitor heavy chain H6 ITIH6 Q6UXX9 R-spondin-2 RSPO2 Q6UY14 ADAMTS-like protein 4 ADAMTSL4 Q6UY27 Prostate and testis expressed protein 2 PATE2 Q6W4X9 Mucin-6 MUC6 Q6WN34 Chordin-like protein 2 CHRDL2 Q6WRI0 Immunoglobulin superfamily member 10 IGSF10 Q6X4U4 Sclerostin domain-containing protein 1 SOSTDC1 Q6X784 Zona pellucida-binding protein 2 ZPBP2 Q6XE38 Secretoglobin family 1D member 4 SCGB1D4 Q6XPR3 Repetin RPTN Q6XZB0 Lipase member I LIPI Q6ZMM2 ADAMTS-like protein 5 ADAMTSL5 Q6ZMP0 Thrombospondin type-1 domain-containing THSD4 protein 4 Q6ZNF0 Iron/zinc purple acid phosphatase-like protein PAPL Q6ZRI0 Otogelin OTOG Q6ZRP7 Sulfhydryl oxidase 2 QSOX2 Q6ZWJ8 Kielin/chordin-like protein KCP Q75N90 Fibrillin-3 FBN3 Q765I0 Urotensin-2B UTS2D Q76B58 Protein FAM5C FAM5C Q76LX8 A disintegrin and metalloproteinase with ADAMTS13 thrombospondin motifs 13 Q76M96 Coiled-coil domain-containing protein 80 CCDC80 Q7L1S5 Carbohydrate sulfotransferase 9 CHST9 Q7L513 Fc receptor-like A FCRLA Q7L8A9 Vasohibin-1 VASH1 Q7RTM1 Otopetrin-1 OTOP1 Q7RTW8 Otoancorin OTOA Q7RTY5 Serine protease 48 PRSS48 Q7RTY7 Ovochymase-1 OVCH1 Q7RTZ1 Ovochymase-2 OVCH2 Q7Z304 MAM domain-containing protein 2 MAMDC2 Q7Z3S9 Notch homolog 2 N-terminal-like protein NOTCH2NL Q7Z4H4 Intermedin-short ADM2 Q7Z4P5 Growth/differentiation factor 7 GDF7 Q7Z4R8 UPF0669 protein C6orf120 C6orf120 Q7Z4W2 Lysozyme-like protein 2 LYZL2 Q7Z5A4 Serine protease 42 PRSS42 Q7Z5A7 Protein FAM19A5 FAM19A5 Q7Z5A8 Protein FAM19A3 FAM19A3 Q7Z5A9 Protein FAM19A1 FAM19A1 Q7Z5J1 Hydroxysteroid 11-beta-dehydrogenase 1-like HSD11B1L protein Q7Z5L0 Vitelline membrane outer layer protein 1 homolog VMO1 Q7Z5L3 Complement C1q-like protein 2 C1QL2 Q7Z5L7 Podocan PODN Q7Z5P4 17-beta-hydroxysteroid dehydrogenase 13 HSD17B13 Q7Z5P9 Mucin-19 MUC19 Q7Z5Y6 Bone morphogenetic protein 8A BMP8A Q7Z7B7 Beta-defensin 132 DEFB132 Q7Z7B8 Beta-defensin 128 DEFB128 Q7Z7C8 Transcription initiation factor TFIID subunit 8 TAF8 Q7Z7H5 Transmembrane emp24 domain-containing protein 4 TMED4 Q86SG7 Lysozyme g-like protein 2 LYG2 Q86SI9 Protein CEI C5orf38 Q86TE4 Leucine zipper protein 2 LUZP2 Q86TH1 ADAMTS-like protein 2 ADAMTSL2 Q86U17 Serpin A11 SERPINA11 Q86UU9 Endokinin-A TAC4 Q86UW8 Hyaluronan and proteoglycan link protein 4 HAPLN4 Q86UX2 Inter-alpha-trypsin inhibitor heavy chain H5 ITIH5 Q86V24 Adiponectin receptor protein 2 ADIPOR2 Q86VB7 Soluble CD163 CD163 Q86VR8 Four-jointed box protein 1 FJX1 Q86WD7 Serpin A9 SERPINA9 Q86WN2 Interferon epsilon IFNE Q86WS3 Placenta-specific 1-like protein PLAC1L Q86X52 Chondroitin sulfate synthase 1 CHSY1 Q86XP6 Gastrokine-2 GKN2 Q86XS5 Angiopoietin-related protein 5 ANGPTL5 Q86Y27 B melanoma antigen 5 BAGE5 Q86Y28 B melanoma antigen 4 BAGE4 Q86Y29 B melanoma antigen 3 BAGE3 Q86Y30 B melanoma antigen 2 BAGE2 Q86Y38 Xylosyltransferase 1 XYLT1 Q86Y78 Ly6/PLAUR domain-containing protein 6 LYPD6 Q86YD3 Transmembrane protein 25 TMEM25 Q86YJ6 Threonine synthase-like 2 THNSL2 Q86YW7 Glycoprotein hormone beta-5 GPHB5 Q86Z23 Complement C1q-like protein 4 C1QL4 Q8IU57 Interleukin-28 receptor subunit alpha IL28RA Q8IUA0 WAP four-disulfide core domain protein 8 WFDC8 Q8IUB2 WAP four-disulfide core domain protein 3 WFDC3 Q8IUB3 Protein WFDC10B WFDC10B Q8IUB5 WAP four-disulfide core domain protein 13 WFDC13 Q8IUH2 Protein CREG2 CREG2 Q8IUK5 Plexin domain-containing protein 1 PLXDC1 Q8IUL8 Cartilage intermediate layer protein 2 C2 CILP2 Q8IUX7 Adipocyte enhancer-binding protein 1 AEBP1 Q8IUX8 Epidermal growth factor-like protein 6 EGFL6 Q8IVL8 Carboxypeptidase O CPO Q8IVN8 Somatomedin-B and thrombospondin type-1 SBSPON domain-containing protein Q8IVW8 Protein spinster homolog 2 SPNS2 Q8IW75 Serpin A12 SERPINA12 Q8IW92 Beta-galactosidase-1-like protein 2 GLB1L2 Q8IWL1 Pulmonary surfactant-associated protein A2 SFTPA2 Q8IWL2 Pulmonary surfactant-associated protein A1 SFTPA1 Q8IWV2 Contactin-4 CNTN4 Q8IWY4 Signal peptide, CUB and EGF-like domain- SCUBE1 containing protein 1 Q8IX30 Signal peptide, CUB and EGF-like domain- SCUBE3 containing protein 3 Q8IXA5 Sperm acrosome membrane-associated protein 3, SPACA3 membrane form Q8IXB1 DnaJ homolog subfamily C member 10 DNAJC10 Q8IXL6 Extracellular serine/threonine protein kinase FAM20C Fam20C Q8IYD9 Lung adenoma susceptibility protein 2 LAS2 Q8IYP2 Serine protease 58 PRSS58 Q8IYS5 Osteoclast-associated immunoglobulin-like OSCAR receptor Q8IZC6 Collagen alpha-1(XXVII) chain COL27A1 Q8IZJ3 C3 and PZP-like alpha-2-macroglobulin domain- CPAMD8 containing protein 8 Q8IZN7 Beta-defensin 107 DEFB107B Q8N0V4 Leucine-rich repeat LGI family member 2 LGI2 Q8N104 Beta-defensin 106 DEFB106B Q8N119 Matrix metalloproteinase-21 MMP21 Q8N129 Protein canopy homolog 4 CNPY4 Q8N135 Leucine-rich repeat LGI family member 4 LGI4 Q8N145 Leucine-rich repeat LGI family member 3 LGI3 Q8N158 Glypican-2 GPC2 Q8N1E2 Lysozyme g-like protein 1 LYG1 Q8N2E2 von Willebrand factor D and EGF domain- VWDE containing protein Q8N2E6 Prosalusin TOR2A Q8N2S1 Latent-transforming growth factor beta-binding LTBP4 protein 4 Q8N302 Angiogenic factor with G patch and FHA domains 1 AGGF1 Q8N307 Mucin-20 MUC20 Q8N323 NXPE family member 1 NXPE1 Q8N387 Mucin-15 MUC15 Q8N3Z0 Inactive serine protease 35 PRSS35 Q8N436 Inactive carboxypeptidase-like protein X2 CPXM2 Q8N474 Secreted frizzled-related protein 1 SFRP1 Q8N475 Follistatin-related protein 5 FSTL5 Q8N4F0 BPI fold-containing family B member 2 BPIFB2 Q8N4T0 Carboxypeptidase A6 CPA6 Q8N5W8 Protein FAM24B FAM24B Q8N687 Beta-defensin 125 DEFB125 Q8N688 Beta-defensin 123 DEFB123 Q8N690 Beta-defensin 119 DEFB119 Q8N6C5 Immunoglobulin superfamily member 1 IGSF1 Q8N6C8 Leukocyte immunoglobulin-like receptor LILRA3 subfamily A member 3 Q8N6G6 ADAMTS-like protein 1 ADAMTSL1 Q8N6Y2 Leucine-rich repeat-containing protein 17 LRRC17 Q8N729 Neuropeptide W-23 NPW Q8N8U9 BMP-binding endothelial regulator protein BMPER Q8N907 DAN domain family member 5 DAND5 Q8NAT1 Glycosyltransferase-like domain-containing GTDC2 protein 2 Q8NAU1 Fibronectin type III domain-containing protein 5 FNDC5 Q8NB37 Parkinson disease 7 domain-containing protein 1 PDDC1 Q8NBI3 Draxin DRAXIN Q8NBM8 Prenylcysteine oxidase-like PCYOX1L Q8NBP7 Proprotein convertase subtilisin/kexin type 9 PCSK9 Q8NBQ5 Estradiol 17-beta-dehydrogenase 11 HSD17B11 Q8NBV8 Synaptotagmin-8 SYT8 Q8NCC3 Group XV phospholipase A2 PLA2G15 Q8NCF0 C-type lectin domain family 18 member C CLEC18C Q8NCW5 NAD(P)H-hydrate epimerase APOA1BP Q8NDA2 Hemicentin-2 HMCN2 Q8NDX9 Lymphocyte antigen 6 complex locus protein G5b LY6G5B Q8NDZ4 Deleted in autism protein 1 C3orf58 Q8NEB7 Acrosin-binding protein ACRBP Q8NES8 Beta-defensin 124 DEFB124 Q8NET1 Beta-defensin 108B DEFB108B Q8NEX5 Protein WFDC9 WFDC9 Q8NEX6 Protein WFDC11 WFDC11 Q8NF86 Serine protease 33 PRSS33 Q8NFM7 Interleukin-17 receptor D IL17RD Q8NFQ5 BPI fold-containing family B member 6 BPIFB6 Q8NFQ6 BPI fold-containing family C protein BPIFC Q8NFU4 Follicular dendritic cell secreted peptide FDCSP Q8NFW1 Collagen alpha-1(XXII) chain COL22A1 Q8NG35 Beta-defensin 105 DEFB105B Q8NG41 Neuropeptide B-23 NPB Q8NHW6 Otospiralin OTOS Q8NI99 Angiopoietin-related protein 6 ANGPTL6 Q8TAA1 Probable ribonuclease 11 RNASE11 Q8TAG5 V-set and transmembrane domain-containing VSTM2A protein 2A Q8TAL6 Fin bud initiation factor homolog FIBIN Q8TAT2 Fibroblast growth factor-binding protein 3 FGFBP3 Q8TAX7 Mucin-7 MUC7 Q8TB22 Spermatogenesis-associated protein 20 SPATA20 Q8TB73 Protein NDNF NDNF Q8TB96 T-cell immunomodulatory protein ITFG1 Q8TC92 Protein disulfide-thiol oxidoreductase ENOX1 Q8TCV5 WAP four-disulfide core domain protein 5 WFDC5 Q8TD06 Anterior gradient protein 3 homolog AGR3 Q8TD33 Secretoglobin family 1C member 1 SCGB1C1 Q8TD46 Cell surface glycoprotein CD200 receptor 1 CD200R1 Q8TDE3 Ribonuclease 8 RNASE8 Q8TDF5 Neuropilin and tolloid-like protein 1 NETO1 Q8TDL5 BPI fold-containing family B member 1 BPIFB1 Q8TE56 A disintegrin and metalloproteinase with ADAMTS17 thrombospondin motifs 17 Q8TE57 A disintegrin and metalloproteinase with ADAMTS16 thrombospondin motifs 16 Q8TE58 A disintegrin and metalloproteinase with ADAMTS15 thrombospondin motifs 15 Q8TE59 A disintegrin and metalloproteinase with ADAMTS19 thrombospondin motifs 19 Q8TE60 A disintegrin and metalloproteinase with ADAMTS18 thrombospondin motifs 18 Q8TE99 Acid phosphatase-like protein 2 ACPL2 Q8TER0 Sushi, nidogen and EGF-like domain-containing SNED1 protein 1 Q8TEU8 WAP, kazal, immunoglobulin, kunitz and NTR WFIKKN2 domain-containing protein 2 Q8WTQ1 Beta-defensin 104 DEFB104B Q8WTR8 Netrin-5 NTN5 Q8WTU2 Scavenger receptor cysteine-rich domain- SRCRB4D containing group B protein Q8WU66 Protein TSPEAR TSPEAR Q8WUA8 Tsukushin TSKU Q8WUF8 Protein FAM172A FAM172A Q8WUJ1 Neuferricin CYB5D2 Q8WUY1 UPF0670 protein THEM6 THEM6 Q8WVN6 Secreted and transmembrane protein 1 SECTM1 Q8WVQ1 Soluble calcium-activated nucleotidase 1 CANT1 Q8WWA0 Intelectin-1 ITLN1 Q8WWG1 Neuregulin-4 NRG4 Q8WWQ2 Inactive heparanase-2 HPSE2 Q8WWU7 Intelectin-2 ITLN2 Q8WWY7 WAP four-disulfide core domain protein 12 WFDC12 Q8WWY8 Lipase member H LIPH Q8WWZ8 Oncoprotein-induced transcript 3 protein OIT3 Q8WX39 Epididymal-specific lipocalin-9 LCN9 Q8WXA2 Prostate and testis expressed protein 1 PATE1 Q8WXD2 Secretogranin-3 SCG3 Q8WXF3 Relaxin-3 A chain RLN3 Q8WXI7 Mucin-16 MUC16 Q8WXQ8 Carboxypeptidase A5 CPA5 Q8WXS8 A disintegrin and metalloproteinase with ADAMTS14 thrombospondin motifs 14 Q92484 Acid sphingomyelinase-like phosphodiesterase 3a SMPDL3A Q92485 Acid sphingomyelinase-like phosphodiesterase 3b SMPDL3B Q92496 Complement factor H-related protein 4 CFHR4 Q92520 Protein FAM3C FAM3C Q92563 Testican-2 SPOCK2 Q92583 C-C motif chemokine 17 CCL17 Q92626 Peroxidasin homolog PXDN Q92743 Serine protease HTRA1 HTRA1 Q92752 Tenascin-R TNR Q92765 Secreted frizzled-related protein 3 FRZB Q92819 Hyaluronan synthase 2 HAS2 Q92820 Gamma-glutamyl hydrolase GGH Q92824 Proprotein convertase subtilisin/kexin type 5 PCSK5 Q92832 Protein kinase C-binding protein NELL1 NELL1 Q92838 Ectodysplasin-A, membrane form EDA Q92874 Deoxyribonuclease-1-like 2 DNASE1L2 Q92876 Kallikrein-6 KLK6 Q92913 Fibroblast growth factor 13 FGF13 Q92954 Proteoglycan 4 C-terminal part PRG4 Q93038 Tumor necrosis factor receptor superfamily TNFRSF25 member 25 Q93091 Ribonuclease K6 RNASE6 Q93097 Protein Wnt-2b WNT2B Q93098 Protein Wnt-8b WNT8B Q95460 Major histocompatibility complex class I-related MR1 gene protein Q969D9 Thymic stromal lymphopoietin TSLP Q969E1 Liver-expressed antimicrobial peptide 2 LEAP2 Q969H8 UPF0556 protein C19orf10 C19orf10 Q969Y0 NXPE family member 3 NXPE3 Q96A54 Adiponectin receptor protein 1 ADIPOR1 Q96A83 Collagen alpha-1(XXVI) chain EMID2 Q96A84 EMI domain-containing protein 1 EMID1 Q96A98 Tuberoinfundibular peptide of 39 residues PTH2 Q96A99 Pentraxin-4 PTX4 Q96BH3 Epididymal sperm-binding protein 1 ELSPBP1 Q96BQ1 Protein FAM3D FAM3D Q96CG8 Collagen triple helix repeat-containing protein 1 CTHRC1 Q96DA0 Zymogen granule protein 16 homolog B ZG16B Q96DN2 von Willebrand factor C and EGF domain- VWCE containing protein Q96DR5 BPI fold-containing family A member 2 BPIFA2 Q96DR8 Mucin-like protein 1 MUCL1 Q96DX4 RING finger and SPRY domain-containing protein 1 RSPRY1 Q96EE4 Coiled-coil domain-containing protein 126 CCDC126 Q96GS6 Abhydrolase domain-containing protein FAM108A1 FAM108A1 Q96GW7 Brevican core protein BCAN Q96HF1 Secreted frizzled-related protein 2 SFRP2 Q96I82 Kazal-type serine protease inhibitor domain- KAZALD1 containing protein 1 Q96ID5 Immunoglobulin superfamily member 21 IGSF21 Q96II8 Leucine-rich repeat and calponin homology LRCH3 domain-containing protein 3 Q96IY4 Carboxypeptidase B2 CPB2 Q96JB6 Lysyl oxidase homolog 4 LOXL4 Q96JK4 HHIP-like protein 1 HHIPL1 Q96KN2 Beta-Ala-His dipeptidase CNDP1 Q96KW9 Protein SPACA7 SPACA7 Q96KX0 Lysozyme-like protein 4 LYZL4 Q96L15 Ecto-ADP-ribosyltransferase 5 ART5 Q96LB8 Peptidoglycan recognition protein 4 PGLYRP4 Q96LB9 Peptidoglycan recognition protein 3 PGLYRP3 Q96LC7 Sialic acid-binding Ig-like lectin 10 SIGLEC10 Q96LR4 Protein FAM19A4 FAM19A4 Q96MK3 Protein FAM20A FAM20A Q96MS3 Glycosyltransferase 1 domain-containing protein 1 GLT1D1 Q96NY8 Processed poliovirus receptor-related protein 4 PVRL4 Q96NZ8 WAP, kazal, immunoglobulin, kunitz and NTR WFIKKN1 domain-containing protein 1 Q96NZ9 Proline-rich acidic protein 1 PRAP1 Q96P44 Collagen alpha-1(XXI) chain COL21A1 Q96PB7 Noelin-3 OLFM3 Q96PC5 Melanoma inhibitory activity protein 2 MIA2 Q96PD5 N-acetylmuramoyl-L-alanine amidase PGLYRP2 Q96PH6 Beta-defensin 118 DEFB118 Q96PL1 Secretoglobin family 3A member 2 SCGB3A2 Q96PL2 Beta-tectorin TECTB Q96QH8 Sperm acrosome-associated protein 5 SPACA5 Q96QR1 Secretoglobin family 3A member 1 SCGB3A1 Q96QU1 Protocadherin-15 PCDH15 Q96QV1 Hedgehog-interacting protein HHIP Q96RW7 Hemicentin-1 HMCN1 Q96S42 Nodal homolog NODAL Q96S86 Hyaluronan and proteoglycan link protein 3 HAPLN3 Q96SL4 Glutathione peroxidase 7 GPX7 Q96SM3 Probable carboxypeptidase X1 CPXM1 Q96T91 Glycoprotein hormone alpha-2 GPHA2 Q99062 Granulocyte colony-stimulating factor receptor CSF3R Q99102 Mucin-4 alpha chain MUC4 Q99217 Amelogenin, X isoform AMELX Q99218 Amelogenin, Y isoform AMELY Q99435 Protein kinase C-binding protein NELL2 NELL2 Q99470 Stromal cell-derived factor 2 SDF2 Q99542 Matrix metalloproteinase-19 MMP19 Q99574 Neuroserpin SERPINI1 Q99584 Protein S100-A13 S100A13 Q99616 C-C motif chemokine 13 CCL13 Q99645 Epiphycan EPYC Q99674 Cell growth regulator with EF hand domain CGREF1 protein 1 Q99715 Collagen alpha-1(XII) chain COL12A1 Q99727 Metalloproteinase inhibitor 4 TIMP4 Q99731 C-C motif chemokine 19 CCL19 Q99748 Neurturin NRTN Q99935 Proline-rich protein 1 PROL1 Q99942 E3 ubiquitin-protein ligase RNF5 RNF5 Q99944 Epidermal growth factor-like protein 8 EGFL8 Q99954 Submaxillary gland androgen-regulated protein 3A SMR3A Q99969 Retinoic acid receptor responder protein 2 RARRES2 Q99972 Myocilin MYOC Q99983 Osteomodulin OMD Q99985 Semaphorin-3C SEMA3C Q99988 Growth/differentiation factor 15 GDF15 Q9BPW4 Apolipoprotein L4 APOL4 Q9BQ08 Resistin-like beta RETNLB Q9BQ16 Testican-3 SPOCK3 Q9BQ51 Programmed cell death 1 ligand 2 PDCD1LG2 Q9BQB4 Sclerostin SOST Q9BQI4 Coiled-coil domain-containing protein 3 CCDC3 Q9BQP9 BPI fold-containing family A member 3 BPIFA3 Q9BQR3 Serine protease 27 PRSS27 Q9BQY6 WAP four-disulfide core domain protein 6 WFDC6 Q9BRR6 ADP-dependent glucokinase ADPGK Q9BS86 Zona pellucida-binding protein 1 ZPBP Q9BSG0 Protease-associated domain-containing protein 1 PRADC1 Q9BSG5 Retbindin RTBDN Q9BT30 Probable alpha-ketoglutarate-dependent ALKBH7 dioxygenase ABH7 Q9BT56 Spexin C12orf39 Q9BT67 NEDD4 family-interacting protein 1 NDFIP1 Q9BTY2 Plasma alpha-L-fucosidase FUCA2 Q9BU40 Chordin-like protein 1 CHRDL1 Q9BUD6 Spondin-2 SPON2 Q9BUN1 Protein MENT MENT Q9BUR5 Apolipoprotein O APOO Q9BV94 ER degradation-enhancing alpha-mannosidase-like 2 EDEM2 Q9BWP8 Collectin-11 COLEC11 Q9BWS9 Chitinase domain-containing protein 1 CHID1 Q9BX67 Junctional adhesion molecule C JAM3 Q9BX93 Group XIIB secretory phospholipase A2-like PLA2G12B protein Q9BXI9 Complement C1q tumor necrosis factor-related C1QTNF6 protein 6 Q9BXJ0 Complement C1q tumor necrosis factor-related C1QTNF5 protein 5 Q9BXJ1 Complement C1q tumor necrosis factor-related C1QTNF1 protein 1 Q9BXJ2 Complement C1q tumor necrosis factor-related C1QTNF7 protein 7 Q9BXJ3 Complement C1q tumor necrosis factor-related C1QTNF4 protein 4 Q9BXJ4 Complement C1q tumor necrosis factor-related C1QTNF3 protein 3 Q9BXJ5 Complement C1q tumor necrosis factor-related C1QTNF2 protein 2 Q9BXN1 Asporin ASPN Q9BXP8 Pappalysin-2 PAPPA2 Q9BXR6 Complement factor H-related protein 5 CFHR5 Q9BXS0 Collagen alpha-1(XXV) chain COL25A1 Q9BXX0 EMILIN-2 EMILIN2 Q9BXY4 R-spondin-3 RSPO3 Q9BY15 EGF-like module-containing mucin-like hormone EMR3 receptor-like 3 subunit beta Q9BY50 Signal peptidase complex catalytic subunit SEC11C SEC11C Q9BY76 Angiopoietin-related protein 4 ANGPTL4 Q9BYF1 Processed angiotensin-converting enzyme 2 ACE2 Q9BYJ0 Fibroblast growth factor-binding protein 2 FGFBP2 Q9BYW3 Beta-defensin 126 DEFB126 Q9BYX4 Interferon-induced helicase C domain-containing IFIH1 protein 1 Q9BYZ8 Regenerating islet-derived protein 4 REG4 Q9BZ76 Contactin-associated protein-like 3 CNTNAP3 Q9BZG9 Ly-6/neurotoxin-like protein 1 LYNX1 Q9BZJ3 Tryptase delta TPSD1 Q9BZM1 Group XIIA secretory phospholipase A2 PLA2G12A Q9BZM2 Group IIF secretory phospholipase A2 PLA2G2F Q9BZM5 NKG2D ligand 2 ULBP2 Q9BZP6 Acidic mammalian chitinase CHIA Q9BZZ2 Sialoadhesin SIGLEC1 Q9C0B6 Protein FAM5B FAM5B Q9GZM7 Tubulointerstitial nephritis antigen-like TINAGL1 Q9GZN4 Brain-specific serine protease 4 PRSS22 Q9GZP0 Platelet-derived growth factor D, receptor- PDGFD binding form Q9GZT5 Protein Wnt-10a WNT10A Q9GZU5 Nyctalopin NYX Q9GZV7 Hyaluronan and proteoglycan link protein 2 HAPLN2 Q9GZV9 Fibroblast growth factor 23 FGF23 Q9GZX9 Twisted gastrulation protein homolog 1 TWSG1 Q9GZZ7 GDNF family receptor alpha-4 GFRA4 Q9GZZ8 Extracellular glycoprotein lacritin LACRT Q9H0B8 Cysteine-rich secretory protein LCCL domain- CRISPLD2 containing 2 Q9H106 Signal-regulatory protein delta SIRPD Q9H114 Cystatin-like 1 CSTL1 Q9H173 Nucleotide exchange factor SIL1 SIL1 Q9H1E1 Ribonuclease 7 RNASE7 Q9H1F0 WAP four-disulfide core domain protein 10A WFDC10A Q9H1J5 Protein Wnt-8a WNT8A Q9H1J7 Protein Wnt-5b WNT5B Q9H1M3 Beta-defensin 129 DEFB129 Q9H1M4 Beta-defensin 127 DEFB127 Q9H1Z8 Augurin C2orf40 Q9H239 Matrix metalloproteinase-28 MMP28 Q9H2A7 C-X-C motif chemokine 16 CXCL16 Q9H2A9 Carbohydrate sulfotransferase 8 CHST8 Q9H2R5 Kallikrein-15 KLK15 Q9H2X0 Chordin CHRD Q9H2X3 C-type lectin domain family 4 member M CLEC4M Q9H306 Matrix metalloproteinase-27 MMP27 Q9H324 A disintegrin and metalloproteinase with ADAMTS10 thrombospondin motifs 10 Q9H336 Cysteine-rich secretory protein LCCL domain- CRISPLD1 containing 1 Q9H3E2 Sorting nexin-25 SNX25 Q9H3R2 Mucin-13 MUC13 Q9H3U7 SPARC-related modular calcium-binding protein 2 SMOC2 Q9H3Y0 Peptidase inhibitor R3HDML R3HDML Q9H4A4 Aminopeptidase B RNPEP Q9H4F8 SPARC-related modular calcium-binding protein 1 SMOC1 Q9H4G1 Cystatin-9-like CST9L Q9H5V8 CUB domain-containing protein 1 CDCP1 Q9H6B9 Epoxide hydrolase 3 EPHX3 Q9H6E4 Coiled-coil domain-containing protein 134 CCDC134 Q9H741 UPF0454 protein C12orf49 C12orf49 Q9H772 Gremlin-2 GREM2 Q9H7Y0 Deleted in autism-related protein 1 CXorf36 Q9H8L6 Multimerin-2 MMRN2 Q9H9S5 Fukutin-related protein FKRP Q9HAT2 Sialate O-acetylesterase SIAE Q9HB40 Retinoid-inducible serine carboxypeptidase SCPEP1 Q9HB63 Netrin-4 NTN4 Q9HBJ0 Placenta-specific protein 1 PLAC1 Q9HC23 Prokineticin-2 PROK2 Q9HC57 WAP four-disulfide core domain protein 1 WFDC1 Q9HC73 Cytokine receptor-like factor 2 CRLF2 Q9HC84 Mucin-5B MUC5B Q9HCB6 Spondin-1 SPON1 Q9HCQ7 Neuropeptide NPSF NPVF Q9HCT0 Fibroblast growth factor 22 FGF22 Q9HD89 Resistin RETN Q9NNX1 Tuftelin TUFT1 Q9NNX6 CD209 antigen CD209 Q9NP55 BPI fold-containing family A member 1 BPIFA1 Q9NP70 Ameloblastin AMBN Q9NP95 Fibroblast growth factor 20 FGF20 Q9NP99 Triggering receptor expressed on myeloid cells 1 TREM1 Q9NPA2 Matrix metalloproteinase-25 MMP25 Q9NPE2 Neugrin NGRN Q9NPH0 Lysophosphatidic acid phosphatase type 6 ACP6 Q9NPH6 Odorant-binding protein 2b OBP2B Q9NQ30 Endothelial cell-specific molecule 1 ESM1 Q9NQ36 Signal peptide, CUB and EGF-like domain- SCUBE2 containing protein 2 Q9NQ38 Serine protease inhibitor Kazal-type 5 SPINK5 Q9NQ76 Matrix extracellular phosphoglycoprotein MEPE Q9NQ79 Cartilage acidic protein 1 CRTAC1 Q9NR16 Scavenger receptor cysteine-rich type 1 CD163L1 protein M160 Q9NR23 Growth/differentiation factor 3 GDF3 Q9NR71 Neutral ceramidase ASAH2 Q9NR99 Matrix-remodeling-associated protein 5 MXRA5 Q9NRA1 Platelet-derived growth factor C PDGFC Q9NRC9 Otoraplin OTOR Q9NRE1 Matrix metalloproteinase-26 MMP26 Q9NRJ3 C-C motif chemokine 28 CCL28 Q9NRM1 Enamelin ENAM Q9NRN5 Olfactomedin-like protein 3 OLFML3 Q9NRR1 Cytokine-like protein 1 CYTL1 Q9NS15 Latent-transforming growth factor beta-binding LTBP3 protein 3 Q9NS62 Thrombospondin type-1 domain-containing THSD1 protein 1 Q9NS71 Gastrokine-1 GKN1 Q9NS98 Semaphorin-3G SEMA3G Q9NSA1 Fibroblast growth factor 21 FGF21 Q9NT22 EMILIN-3 EMILIN3 Q9NTU7 Cerebellin-4 CBLN4 Q9NVR0 Kelch-like protein 11 KLHL11 Q9NWH7 Spermatogenesis-associated protein 6 SPATA6 Q9NXC2 Glucose-fructose oxidoreductase domain- GFOD1 containing protein 1 Q9NY56 Odorant-binding protein 2a OBP2A Q9NY84 Vascular non-inflammatory molecule 3 VNN3 Q9NZ20 Group 3 secretory phospholipase A2 PLA2G3 Q9NZC2 Triggering receptor expressed on myeloid cells 2 TREM2 Q9NZK5 Adenosine deaminase CECR1 CECR1 Q9NZK7 Group IIE secretory phospholipase A2 PLA2G2E Q9NZP8 Complement C1r subcomponent-like protein C1RL Q9NZV1 Cysteine-rich motor neuron 1 protein CRIM1 Q9NZW4 Dentin sialoprotein DSPP Q9P0G3 Kallikrein-14 KLK14 Q9P0W0 Interferon kappa IFNK Q9P218 Collagen alpha-1(XX) chain COL20A1 Q9P2C4 Transmembrane protein 181 TMEM181 Q9P2K2 Thioredoxin domain-containing protein 16 TXNDC16 Q9P2N4 A disintegrin and metalloproteinase with ADAMTS9 thrombospondin motifs 9 Q9UBC7 Galanin-like peptide GALP Q9UBD3 Cytokine SCM-1 beta XCL2 Q9UBD9 Cardiotrophin-like cytokine factor 1 CLCF1 Q9UBM4 Opticin OPTC Q9UBP4 Dickkopf-related protein 3 DKK3 Q9UBQ6 Exostosin-like 2 EXTL2 Q9UBR5 Chemokine-like factor CKLF Q9UBS5 Gamma-aminobutyric acid type B receptor subunit 1 GABBR1 Q9UBT3 Dickkopf-related protein 4 short form DKK4 Q9UBU2 Dickkopf-related protein 2 DKK2 Q9UBU3 Ghrelin-28 GHRL Q9UBV4 Protein Wnt-16 WNT16 Q9UBX5 Fibulin-5 FBLN5 Q9UBX7 Kallikrein-11 KLK11 Q9UEF7 Klotho KL Q9UFP1 Protein FAM198A FAM198A Q9UGM3 Deleted in malignant brain tumors 1 protein DMBT1 Q9UGM5 Fetuin-B FETUB Q9UGP8 Translocation protein SEC63 homolog SEC63 Q9UHF0 Neurokinin-B TAC3 Q9UHF1 Epidermal growth factor-like protein 7 EGFL7 Q9UHG2 ProSAAS PCSK1N Q9UHI8 A disintegrin and metalloproteinase with ADAMTS1 thrombospondin motifs 1 Q9UHL4 Dipeptidyl peptidase 2 DPP7 Q9UI42 Carboxypeptidase A4 CPA4 Q9UIG4 Psoriasis susceptibility 1 candidate gene 2 protein PSORS1C2 Q9UIK5 Tomoregulin-2 TMEFF2 Q9UIQ6 Leucyl-cystinyl aminopeptidase, pregnancy serum LNPEP form Q9UJA9 Ectonucleotide pyrophosphatase/phosphodiesterase ENPP5 family member 5 Q9UJH8 Meteorin METRN Q9UJJ9 N-acetylglucosamine-1-phosphotransferase GNPTG subunit gamma Q9UJW2 Tubulointerstitial nephritis antigen TINAG Q9UK05 Growth/differentiation factor 2 GDF2 Q9UK55 Protein Z-dependent protease inhibitor SERPINA10 Q9UK85 Dickkopf-like protein 1 DKKL1 Q9UKJ1 Paired immunoglobulin-like type 2 receptor alpha PILRA Q9UKP4 A disintegrin and metalloproteinase with ADAMTS7 thrombospondin motifs 7 Q9UKP5 A disintegrin and metalloproteinase with ADAMTS6 thrombospondin motifs 6 Q9UKQ2 Disintegrin and metalloproteinase domain- ADAM28 containing protein 28 Q9UKQ9 Kallikrein-9 KLK9 Q9UKR0 Kallikrein-12 KLK12 Q9UKR3 Kallikrein-13 KLK13 Q9UKU9 Angiopoietin-related protein 2 ANGPTL2 Q9UKZ9 Procollagen C-endopeptidase enhancer 2 PCOLCE2 Q9UL52 Transmembrane protease serine 11E non- TMPRSS11E catalytic chain Q9ULC0 Endomucin EMCN Q9ULI3 Protein HEG homolog 1 HEG1 Q9ULZ1 Apelin-13 APLN Q9ULZ9 Matrix metalloproteinase-17 MMP17 Q9UM21 Alpha-1,3-mannosyl-glycoprotein 4-beta-N- MGAT4A acetylglucosaminyltransferase A soluble form Q9UM22 Mammalian ependymin-related protein 1 EPDR1 Q9UM73 ALK tyrosine kinase receptor ALK Q9UMD9 97 kDa linear IgA disease antigen COL17A1 Q9UMX5 Neudesin NENF Q9UN73 Protocadherin alpha-6 PCDHA6 Q9UNA0 A disintegrin and metalloproteinase with ADAMTS5 thrombospondin motifs 5 Q9UNI1 Chymotrypsin-like elastase family member 1 CELA1 Q9UNK4 Group IID secretory phospholipase A2 PLA2G2D Q9UP79 A disintegrin and metalloproteinase with ADAMTS8 thrombospondin motifs 8 Q9UPZ6 Thrombospondin type-1 domain-containing THSD7A protein 7A Q9UQ72 Pregnancy-specific beta-1-glycoprotein 11 PSG11 Q9UQ74 Pregnancy-specific beta-1-glycoprotein 8 PSG8 Q9UQC9 Calcium-activated chloride channel regulator 2 CLCA2 Q9UQE7 Structural maintenance of chromosomes protein 3 SMC3 Q9UQP3 Tenascin-N TNN Q9Y223 UDP-N-acetylglucosamine 2-epimerase GNE Q9Y240 C-type lectin domain family 11 member A CLEC11A Q9Y251 Heparanase 8 kDa subunit HPSE Q9Y258 C-C motif chemokine 26 CCL26 Q9Y264 Angiopoietin-4 ANGPT4 Q9Y275 Tumor necrosis factor ligand superfamily member TNFSF13B 13b, membrane form Q9Y287 BRI2 intracellular domain ITM2B Q9Y2E5 Epididymis-specific alpha-mannosidase MAN2B2 Q9Y334 von Willebrand factor A domain-containing VWA7 protein 7 Q9Y337 Kallikrein-5 KLK5 Q9Y3B3 Transmembrane emp24 domain-containing protein 7 TMED7 Q9Y3E2 BolA-like protein 1 BOLA1 Q9Y426 C2 domain-containing protein 2 C2CD2 Q9Y4K0 Lysyl oxidase homolog 2 LOXL2 Q9Y4X3 C-C motif chemokine 27 CCL27 Q9Y5C1 Angiopoietin-related protein 3 ANGPTL3 Q9Y5I2 Protocadherin alpha-10 PCDHA10 Q9Y5I3 Protocadherin alpha-1 PCDHA1 Q9Y5K2 Kallikrein-4 KLK4 Q9Y5L2 Hypoxia-inducible lipid droplet-associated protein HILPDA Q9Y5Q5 Atrial natriuretic peptide-converting enzyme CORIN Q9Y5R2 Matrix metalloproteinase-24 MMP24 Q9Y5U5 Tumor necrosis factor receptor superfamily TNFRSF18 member 18 Q9Y5W5 Wnt inhibitory factor 1 WIF1 Q9Y5X9 Endothelial lipase LIPG Q9Y625 Secreted glypican-6 GPC6 Q9Y646 Carboxypeptidase Q CPQ Q9Y6C2 EMILIN-1 EMILIN1 Q9Y6F9 Protein Wnt-6 WNT6 Q9Y6I9 Testis-expressed sequence 264 protein TEX264 Q9Y6L7 Tolloid-like protein 2 TLL2 Q9Y6N3 Calcium-activated chloride channel regulator CLCA3P family member 3 Q9Y6N6 Laminin subunit gamma-3 LAMC3 Q9Y6R7 IgGFc-binding protein FCGBP Q9Y6Y9 Lymphocyte antigen 96 LY96 Q9Y6Z7 Collectin-10 COLEC10

In some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more additional exemplary proteins listed in Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 2 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from the proteins listed in Table 2 (or a homolog thereof) along with other components set out herein.

TABLE 2 Additional Exemplary Proteins Uniprot ID Protein Name Gene Name A6NGW2 Putative stereocilin-like protein STRCP1 A6NIE9 Putative serine protease 29 PRSS29P A6NJ16 Putative V-set and immunoglobulin IGHV4OR15-8 domain-containing-like protein IGHV4OR15-8 A6NJS3 Putative V-set and immunoglobulin IGHV1OR21-1 domain-containing-like protein IGHV1OR21-1 A6NMY6 Putative annexin A2-like protein ANXA2P2 A8MT79 Putative zinc-alpha-2-glycoprotein-like 1 A8MWS1 Putative killer cell immunoglobulin-like KIR3DP1 receptor like protein KIR3DP1 A8MXU0 Putative beta-defensin 108A DEFB108P1 C9JUS6 Putative adrenomedullin-5-like protein ADM5 P0C7V7 Putative signal peptidase complex SEC11B catalytic subunit SEC11B P0C854 Putative cat eye syndrome critical region CECR9 protein 9 Q13046 Putative pregnancy-specific beta-1- PSG7 glycoprotein 7 Q16609 Putative apolipoprotein(a)-like protein 2 LPAL2 Q2TV78 Putative macrophage-stimulating protein MST1P9 MSTP9 Q5JQD4 Putative peptide YY-3 PYY3 Q5R387 Putative inactive group IIC secretory PLA2G2C phospholipase A2 Q5VSP4 Putative lipocalin 1-like protein 1 LCN1P1 Q5W188 Putative cystatin-9-like protein CST9LP1 CST9LP1 Q6UXR4 Putative serpin A13 SERPINA13P Q86SH4 Putative testis-specific prion protein PRNT Q86YQ2 Putative latherin LATH Q8IVG9 Putative humanin peptide MT-RNR2 Q8NHM4 Putative trypsin-6 TRY6 Q8NHW4 C-C motif chemokine 4-like CCL4L2 Q9H7L2 Putative killer cell immunoglobulin-like KIR3DX1 receptor-like protein KIR3DX1 Q9NRI6 Putative peptide YY-2 PYY2 Q9UF72 Putative TP73 antisense gene protein 1 TP73-AS1 Q9UKY3 Putative inactive carboxylesterase 4 CES1P1

The Uniprot IDs set forth in Table 1 and Table 2 refer to the human versions the listed proteins and the sequences of each are available from the Uniprot database. Sequences of the listed proteins are also generally available for various animals, including various mammals and animals of veterinary or industrial interest. Accordingly, in some embodiments, compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more proteins chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of the secreted proteins listed in Table 1 or Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 1 or Table 2 along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 1 or Table 2 along with other components set out herein. In some embodiments, mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homologs. In some embodiments, the animal of veterinary or industrial interest is chosen from the mammals listed above and/or chicken, duck, turkey, salmon, catfish, or tilapia.

In embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding a lysosomal protein chosen from Table 3. In some embodiments, the compositions and methods of the invention provide for the delivery of one or more mRNAs encoding one or more lysosomal and/or related proteins listed in Table 3; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 3 (or a homolog thereof) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein chosen from the proteins listed in Table 3 (or a homolog thereof) along with other components set out herein.

TABLE 3 Lysosomal and Related Proteins α-fucosidase α-galactosidase α-glucosidase α-Iduronidase α-mannosidase α-N-acetylgalactosaminidase (α-galactosidase B) β-galactosidase β-glucuronidase β-hexosaminidase β-mannosidase 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase 3-methylcrotonyl-CoA carboxylase 3-O-sulfogalactosyl cerebroside sulfatase (arylsulfatase A) acetyl-CoA transferase acid alpha-glucosidase acid ceramidase acid lipase acid phosphatase acid sphingomyelinase alpha-galactosidase A arylsulfatase A beta-galactosidase beta-glucocerebrosidase beta-hexosaminidase biotinidase cathepsin A cathepsin K CLN3 CLN5 CLN6 CLN8 CLN9 cystine transporter (cystinosin) cytosolic protein beta3A subunit of the adaptor protein-3 complex, AP3 formyl-Glycine generating enzyme (FGE) galactocerebrosidase galactose-1-phosphate uridyltransferase (GALT) galactose 6-sulfate sulfatase (also known as N-acetylgalactosamine-6-sulfatase) glucocerebrosidase glucuronate sulfatase glucuronidase glycoprotein cleaving enzymes glycosaminoglycan cleaving enzymes glycosylasparaginase (aspartylglucosaminidase) GM2-AP Heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT, TMEM76) Heparan sulfatase hexosaminidase A lysosomal proteases methylmalonyl-CoA mutase hyaluronidase Iduronate sulfatase LAMP-2 lysosomal α-mannosidase Lysosomal p40 (C2orf18) Major facilitator superfamily domain containing 8 protein (MFSD8 or CLN7) N-acetylgalactosamine 4-sulfatase N-acetyl glucosamine 6-sulfatase N-acetyl glucosaminidase N-acetylglucosamine-1-phosphate transferase NPC1 NPC2 palmitoyl-protein thioesterase palmitoyl-protein thioesterase (CLN1) Saposin A (Sphingolipid activator protein A) Saposin B (Sphingolipid activator protein B) Saposin C (Sphingolipid activator protein C) Saposin D (Sphingolipid activator protein D) sialic acid transporter (sialin) sialidase Sialin sulfatase Transmembrane protein 74 (TMEM74) tripeptidyl-peptidase tripeptidyl-peptidase I (CLN2) UDP-N-acetylglucosamine-phosphotransferase

Information regarding lysosomal proteins is available from Lubke et al., “Proteomics of the Lysosome,” Biochim Biophys Acta. (2009) 1793: 625-635. In some embodiments, the protein listed in Table 3 and encoded by mRNA in the compositions and methods of the invention is a human protein. Sequences of the listed proteins are also available for various animals, including various mammals and animals of veterinary or industrial interest as described above.

In some embodiments, the compositions and methods of the invention provide for the delivery of mRNA encoding a therapeutic protein (e.g., cytosolic, transmembrane or secreted) such as those listed in Table 4. In some embodiments, the compositions and methods of the invention provide for the delivery of an mRNA encoding a therapeutic protein useful in treating a disease or disorder (i.e., indication) listed in Table 4; thus, compositions of the invention may comprise an mRNA encoding a therapeutic protein listed or not listed in Table 4 (or a homolog thereof, as discussed below) along with other components set out herein for treating a disease or disorder (i.e., indication) listed in Table 4, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a such a protein (or a homolog thereof, as discussed below) along with other components set out herein for treatment of a disease or disorder listed in Table 4.

TABLE 4 Exemplary Indications and Related Proteins Indication Therapeutic Protein 3-Methylcrotonyl-CoA carboxylase deficiency Methylcrotonoyl-CoA carboxylase 3-Methylglutaconic aciduria Methylglutaconyl-CoA hydratase Actinic keratosis Acute intermittent porphyria Porphobilinogen deaminase Acute lymphocytic leukemia Acute myeloid leukemia Addison's disease Adenosine deaminase deficiency Adenosine deaminase Adrenoleukodystrophy ABCD1 Adrenomyeloneuropathy AIDS/HIV Alcohol use disorders Alkaptonuria Homogentisate 1,2-dioxygenase Allergic asthma Anti-IgE mAb Allergies (dermatitis, rhinitis) Alopecia areata Alpers' disease POLG Alpers-Huttenlocher syndrome Alpha 1-antitrypsin deficiency Alpha 1 protease inhibitor Alpha-mannosidosis Alpha-D-mannosidase Alport syndrome Alzheimer's disease Amyloid light-chain amyloidosis Amyotrophic lateral sclerosis (ALS) Anemia Erythropoietin Aortic valve stenosis Argininemia Arginase Argininosuccinic acidemia Argininosuccinate lyase Arrhythmogenic right ventricular dysplasia Autism Autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions Autosomal recessive polycystic kidney disease ARPKD Bacterial infections Basal cell carcinoma Batten disease Battenin + others B-cell chronic lymphocytic leukemia Becker muscular dystrophy Dystrophin Beta-thalassemia Beta globin Binge eating disorder Bipolar disorder Bladder cancer Blepharospasm, Cervical dystonia, Chronic migraine, Botulinum toxin more Bronchiolitis obliterans Brugada syndrome Buerger's disease CACNA1A CACNB4-related Episodic Ataxia Type 2 Cancer and depression Cancer and sexual dysfunction Cancer in pregnancy Carbamylphosphate synthetase deficiency Carbamylphosphate synthetase Carcinoma of the gallbladder Cardiomyopathy (diabetic) Cardiomyopathy (hypertrophic) Carnitine uptake defect SLC22A5 Catecholaminergic polymorphic ventricular tachycardia CDKL5-related Atypical Rett Syndrome Celiac disease Cellulitis Cerebrovascular disease Cervix uteri cancer Chronic fatigue syndrome Chronic graft versus host disease Chronic idiopathic urticaria Chronic immune thrombocytopenia Thrombopoietin Chronic kidney kisease Chronic liver disease Chronic lymphocytic leukemia Chronic myeloid leukemia Chronic pancreatitis Cirrhosis of the liver Citrullinemia, type I Argininosuccinate synthase Classic Rett Syndrome Classical galactosemia Galactose-1-phosphate uridylyltransferase Clostridium difficile associated diarrhea Clotting disorders COAD/COPD Cocaine addiction COL4A5-related disorders Cold contact urticaria Contraception, female Coronary artery diseases Corpus uteri cancer Corticobasal degeneration Crigler-Najjar syndrome UDP-glucuronosyltransferase Critical limb ischemia CTNS-related cystinosis Cutaneous lupus erythematosus Cutaneous neuroendocrine carcinoma (Merkel Cell) Cystic fibrosis CFTR Cystic fibrosis Deoxyribonuclease I Cystinosis Cystinosin Cystinuria SLC7A9 Dementia (Lewy body) Depression Diabetic foot infections Diabetic foot ulcer Diabetic peripheral neuropathy Diabetic ulcers Diarrhoeal diseases Diffuse large B-cell lymphoma DiGeorge syndrome Diverticulitis Drug use disorders Duchenne muscular dystrophy Dystrophin Dysarthria Dyskinesia (levodopa-induced) Early-onset autosomal dominant Alzheimer's disease Eczema Ehlers-Danlos syndrome, type 1 EIF2B1 EIF2B2 EIF2B3 EIF2B4 EIF2B5-related childhood ataxia with central nervous system hypomyelination/vanishing white matter Eosinophilic esophagitis Epilepsy Erectile dysfunction Erythropoietic protoporphyria Ferrochelatase Esophageal carcinoma Essential tremor Fabry disease Alpha galactosidase Familial adenomatous polyposis APC Familial chylomicronemia Lipoprotein lipase Familial dysbetalipoproteinemia Apolipoprotein E Familial isolated dilated cardiomyopathy Familial mediterranean fever Pyrin (MEFV) Familial melanoma Female infertility Follicle stimulating hormone Female sexual dysfunction Fibromyalgia FMR1-related disorders Fracture healing Fragile X Premature Ovarian Failure Syndrome Fragile X syndrome FMRP Fragile X-Associated Tremor/Ataxia Syndrome Friedreich's ataxia Frontotemporal dementia Fryns syndrome Galactocerebrosidase deficiencies GALE deficiency Galactose epimerase GALK deficiency Galactokinase GALT-related galactosemia Gastric cancer Gastroesophageal reflux disease Gaucher disease Glucocerebrosidase Gilbert syndrome UDP-glucuronosyltransferase Glioblastoma multiforme Glomerulonephritis Glutaric acidemia, type I Glutaryl-CoA dehydrogenase GM2 gangliosidosis HEXA, HEXB Gout Urate oxidase Graft versus host disease Growth hormone deficiency Growth hormone 1/Growth hormone 2 Head and neck cancer, Metastatic colorectal cancer Anti-EGFr mAb Hearing loss, adult onset Heart failure Hemachromatosis HFE protein Hemifacial spasm Hemolytic uremic syndrome Anti-complement factor C5 mAb Hemophilia A Factor VIII Hemophilia A, Hemophilia B Factor VII Hemophilia B Factor IX Hepatitis B, Hepatitis C Interferon alpha HER2+ breast cancer, gastric cancer Anti-HER2 mAb Hereditary angioedema C1 esterase inhibitor Hereditary hemorrhagic telangiectasia Hereditary hemorrhagic telangiectasia (AT) Hereditary spherocytosis Hidradenitis suppurativa Homocystinuria Cystathionine beta-synthase Homozygous familial hypercholesterolemia LDL receptor Hunter syndrome (MPS II) Iduronate-2-sulfatase Huntington disease Huntingtin Hurler syndrome (MPS I) Alpha-L iduronidase Hydrolethalus Hyperalgesia Hyperbilirubinemia Hyperhidrosis Hyperlipidemia Hypermethioninemia Methionine adenosyltransferase Hyperoxaluria, type I Serine-pyruvate aminotransferase Hypertension Hyperuricemia Hyponatremia Hypoparathyroidism Parathyroid hormone Hypophosphatasia TNSALP Idiopathic pulmonary fibrosis Iminoglycinuria Immunoglobulin deficiency Immunoglobulin Infection (adenovirus) Infection (anthrax prophylaxis) Infection (BK virus) Infection (Clostridium difficile prophylaxis) Infection (Dengue fever prophylaxis) Infection (Epstein-Barr virus) Infection (Hepatitis-D) Infection (Lyme disease prophylaxis) Infection (Smallpox virus) Infectious diseases vaccines Infectious antigen Inflammatory heart diseases Insomnia Interstitial cystitis Iron-deficiency anaemia Irritable bowel disease Ischaemic heart disease Isovaleric aciduria Isovaleric acid CoA dehydrogenase deficiency Jansky-Bielschowsky disease Juvenile Batten disease Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) Juvenile rheumatoid arthritis TNF-alpha inhibitors Kennedy's disease (SBMA) Keratoconus Krabbe disease Galactocerebrosidase Leber's hereditary optic neuropathy NADH dehydrogenase Leiomyosarcoma Lennox-Gastaut syndrome Lesch-Nyhan syndrome Hypoxanthine phosphoribosyltransferase 1 Leukaemia Li-Fraumeni syndrome TP53 Lipoma Liposarcoma Liver cancer Long-chain 3-OH acyl-CoA dehydrogenase deficiency Long-chain-3-hydroxyacyl-CoA dehydrogenase Lower respiratory infections Lysosomal acid lipase deficiency Lysosomal acid lipase Macular degeneration Major depressive disorder Malignant fibrous histiocytoma Mantle cell lymphoma Maple syrup urine disease 3-methyl-2-oxobutanoate dehydrogenase Marfan syndrome FBN1 Maroteaux-Lamy syndrome (MPS VI) N-acetylgalactosamine 4-sulfatase Mastocytosis McArdle disease Muscle glycogen phosphorylase MECP2-related disorders MECP2-related Severe Neonatal Encephalopathy Medium-chain acyl-CoA dehydrogenase deficiency Acyl-CoA dehydrogenase Melanoma Anti-CTLA4 mAb Metachromatic leukodystrophy Arylsulfatase A Metastatic colorectal cancer, NSCLC, others Anti-VEGF mAb Methylmalonyl-CoA mutase deficiency Methylmalonyl-CoA mutase Migraine Mitochondrial oxidative phosphorylation disorders Morquio syndrome, type A (MPS IVA) Galactose 6-sulfate sulfatase Morquio syndrome, type B (MPS IVB) Beta-galactosidase Mouth and oropharynx cancers Multiple carboxylase deficiency Biotin-methylcrotonoyl-CoA-carboxylase ligase Multiple myeloma Multiple sclerosis Anti-VLA-4 mAb Multiple sclerosis Interferon beta Multiple system atrophy Myasthenia gravis Myelofibrosis Narcolepsy Neonatal bronchopulmonary dysplasia Neonatal infections Nephritis and nephrosis Neurofibromatosis, type 1 NF-1 Neuronal ceroid lipofuscinoses-related diseases Neutropenia G-CSF Niemann Pick disease, type A/B SMPD1 Niemann Pick disease, type C NPC1 Niemann-Pick disease Type C1 Nocturia Non-alcoholic fatty liver disease Non-Hodgkin lymphoma Anti-CD20 mAb Non-small cell lung cancer Notch-3 related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) Obesity Ophthalmoparesis Opioid induced constipation Ornithine transcarbamylase deficiency Ornithine transcarbamylase Osteoarthritis Osteopetrosis Osteoporosis Anti-RANKL mAb Ovarian cancer Paget disease of bone Sequestosome 1 Pain Pancreatic carcinoma Panic disorder Parkinson disease Paroxysmal nocturnal hemoglobinuria Anti-complement factor C5 Mab Pediculosis capitis (head lice) Pelizaeus-Merzbacher disease Pemphigus vulgaris Peptic ulcer disease Peripheral neuropathy Peyronie's disease Phenylketonuria Phenylalanine hydroxylase Pneumococcal infection prophylaxis POLG-related sensory ataxic neuropathy Polycystic kidney disease Polycystic ovary syndrome Polycythaemia vera Polymerase G-related disorders Polymorphous light eruption Pompe disease Alpha glucosidase Porphyria cutanea tarda Uroporphyrinogen decarboxylase Post herpetic neuralgia Post-organ transplant Pouchitis PPM-X Syndrome Prader-Willi syndrome Preeclampsia Premature ejaculation Prematurity and low birth weight Primary ciliary dyskinesia Primary glomerular diseases Primary humoral immune deficiencies (e.g., CVID) Immunoglobulin Proctitis Progressive multifocal leukoencephalopathy Progressive supranuclear palsy Propionic acidemia Propionyl-CoA carboxylase Prostate cancer Psoriasis Anti-IL-12 & IL-23 mAb Psoriatic arthritis TNF-alpha inhibitors PTT-1 Pulmonary arterial hypertension Pulmonary arterial hypertension Raynaud's phenomenon Refractive errors Renal cell carcinoma Restless leg syndrome Retinitis pigmentosa Rheumatic heart disease Rheumatoid arthritis Anti-interleukin-6 (IL-6) mAb Rheumatoid arthritis T-cell costimulation blocker Rheumatoid arthritis TNF-alpha inhibitor Romano-Ward syndrome Rosacea Sanfilippo syndrome, type A (MPS IIIA) Heparan N-sulfatase Sanfilippo syndrome, type B (MPS IIIB) N-acetyl-alpha-D-glucosaminidase Santavuori-Haltia disease Schizophrenia Schnitzler syndrome Scleroderma SCN1A SCN1B-related seizure disorders Short-chain acyl-CoA dehydrogenase deficiency Butyryl-CoA dehydrogenase Sickle cell disease Hemoglobin SLC3A1-related disorders Small cell lung cancer SMN-1-related spinal muscular atrophy (SMA) Spinal muscular atrophy Survival motor neuron protein Squamous cell carcinoma of head and neck Stickler syndrome Stomach cancer Stroke prophylaxis Synovial sarcoma Systemic lupus erythematosus Anti-BAFF Systemic sclerosis Tetrahydrobiopterin-deficient hyperphenylalaninemia Tetrahydrobiopterin Thromboangiitis obliterans Thrombotic disorders Thyroid cancer TPP1 deficiencies Trachea, bronchus, lung cancers Tricuspid atresia TSC1 TSC2-related tuberous sclerosis Type 2 diabetes mellitus Glucagon-like peptide 1 (GLP-1) agonist Type 2 diabetes mellitus Insulin Tyrosinemia, type I Fumarylacetoacetase Ulcerative colitis Uterine fibroids Varicose veins Venous thromboembolism Very long-chain acyl-CoA dehydrogenase deficiency Long-chain-acyl-CoA dehydrogenase von Gierke's disease Glucose-6-phosphatase Von Hippel-Lindau disease pVHL Wegener granulomatosis Wilson disease Wilson disease protein X-Linked adrenal hypoplasia X-linked adrenoleukodystrophy X-linked agammaglobulinemia Bruton's tyrosine kinase

In some embodiments, the present invention is used to prevent, treat and/or cure a subject affected with a disease or disorder listed or associated with the proteins listed in Tables 1, 2, 3 or 4. In some embodiments, an mRNA encodes one or more of argininosuccinate synthetase (ASS1), Factor IX, survival motor neuron 1 (SMN1), or phenylalanine hydroxylase Synthesis of mRNA

mRNAs according to the present invention may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.

In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.

Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.

Modified mRNA

In some embodiments, mRNA according to the present invention may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. An modified mRNA according to the invention can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, .beta.-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5′-O-(1-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine 5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate), 2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine 5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate), 2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide (2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate), 2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine 5′-triphosphate, 2′-arauridine 5′-triphosphate), or azidotriphosphates (2′-azido-2′-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine 5′-triphosphate).

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine 5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine 5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate, 5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate, 5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate, 5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate, 5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate, 6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate, 7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate, 8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate, benzimidazole riboside 5′-triphosphate, N1-methyladenosine 5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine 5′-triphosphate, 06-methylguanosine 5′-triphosphate, pseudouridine 5′-triphosphate, puromycin 5′-triphosphate or xanthosine 5′-triphosphate.

Cap Structure

Typically, mRNA synthesis includes the addition of a “cap” on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a 5′ cap structure. A 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

In some embodiments, naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m⁷G(5′)ppp(5′)N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5′ terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.

A common cap for mRNA produced by in vitro transcription is m⁷G(5′)ppp(5′)G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5′-termini. The prevailing method for the in vitro synthesis of capped mRNA employs a pre-formed dinucleotide of the form m⁷G(5′)ppp(5′)G (“m⁷GpppG”) as an initiator of transcription.

To date, a usual form of a synthetic dinucleotide cap used in in vitro translation experiments is the Anti-Reverse Cap Analog (“ARCA”) or modified ARCA, which is generally a modified cap analog in which the 2′ or 3′ OH group is replaced with —OCH₃.

Additional cap analogs include, but are not limited to, chemical structures selected from the group consisting of m⁷GpppG, m⁷GpppA, m⁷GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m^(2,7)GpppG), trimethylated cap analog (e.g., m^(2,2,7)GpppG), dimethylated symmetrical cap analogs (e.g., m⁷Gpppm⁷G), or anti reverse cap analogs (e.g., ARCA; m^(7,2′Ome)GpppG, m^(72′d)GpppG, m^(7,3′Ome)GpppG, m^(7,3′d)GpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).

In some embodiments, a suitable cap is a 7-methyl guanylate (“m⁷G”) linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in m⁷G(5′)ppp(5′)N, where N is any nucleoside. A preferred embodiment of a m⁷G cap utilized in embodiments of the invention is m⁷G(5′)ppp(5′)G.

In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a 2′-O-methyl residue of the ribose attached to bases 1 and 2. In some embodiments, the cap is a Cap1 structure. Cap1 structures have a 2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. Cap2 structures have a 2′-O-methyl residue attached to both bases 2 and 3.

A variety of m⁷G cap analogs are known in the art, many of which are commercially available. These include the m⁷GpppG described above, as well as the ARCA 3′-OCH₃ and 2′-OCH₃ cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodiments of the invention include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)), phosphorothioate cap analogs (described in Grudzien-Nogalska, E., et al., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylated cap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529, incorporated by reference herein.

Tail Structure

Typically, the presence of a “tail” serves to protect the mRNA from exonuclease degradation. The poly A tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A tail can be added to an mRNA molecule thus rendering the RNA more stable. Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. Poly A may also be ligated to the 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a 3′ poly(A) tail structure. Typically, the length of the poly A tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides (SEQ ID NO: 12). In some embodiments, a poly-A tail on the 3′ terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (SEQ ID NO: 13) (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3′ poly(C) tail structure. A suitable poly-C tail on the 3′ terminus of mRNA typically include about 10 to 200 cytosine nucleotides (SEQ ID NO: 14) (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.

In some embodiments, the length of the poly A or poly C tail is adjusted to control the stability of a modified sense mRNA molecule of the invention and, thus, the transcription of protein. For example, since the length of the poly A tail can influence the half-life of a sense mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and/or polypeptide production in a target cell.

′ and 3′ Untranslated Region

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region. In some embodiments, a 5′ untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5′ untranslated region may be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3′ untranslated region may be between 50 and 500 nucleotides in length or longer.

Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, a 5′ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof to the 3′ end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.

According to various embodiments, any size mRNA may be encapsulated by provided liposomes. In some embodiments, the provided liposomes may encapsulate mRNA of greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.

Formation of Liposomes

The liposomes for use in provided compositions can be prepared by various techniques which are presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then added to the vessel with a vortexing motion which results in the formation of MLVs. Uni-lamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multi-lamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

In certain embodiments, provided compositions comprise a liposome wherein the mRNA is associated on both the surface of the liposome and encapsulated within the same liposome. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the mRNA through electrostatic interactions. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the mRNA through electrostatic interactions.

In some embodiments, the compositions and methods of the invention comprise mRNA encapsulated in a liposome. In some embodiments, the one or more mRNA species may be encapsulated in the same liposome. In some embodiments, the one or more mRNA species may be encapsulated in different liposomes. In some embodiments, the mRNA is encapsulated in one or more liposomes, which differ in their lipid composition, molar ratio of lipid components, size, charge (Zeta potential), targeting ligands and/or combinations thereof. In some embodiments, the one or more liposome may have a different composition of cationic lipids, neutral lipid, PEG-modified lipid and/or combinations thereof. In some embodiments the one or more lipisomes may have a different molar ratio of cationic lipid, neutral lipid, cholesterol and PEG-modified lipid used to create the liposome.

The process of incorporation of a desired mRNA into a liposome is often referred to as “loading”. Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference. The liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane. The incorporation of a nucleic acid into liposomes is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the liposome. The purpose of incorporating a mRNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and/or facilitate the delivery of mRNA to the target cell or tissue.

Liposome Size

Suitable liposomes in accordance with the present invention may be made in various sizes. In some embodiments, provided liposomes may be made smaller than previously known mRNA encapsulating liposomes. In some embodiments, decreased size of liposomes is associated with more efficient delivery of mRNA. Selection of an appropriate liposome size may take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made.

In some embodiments, an appropriate size of liposome is selected to facilitate systemic distribution of antibody encoded by the mRNA. In some embodiments, it may be desirable to limit transfection of the mRNA to certain cells or tissues. For example, to target hepatocytes a liposome may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; in such cases the liposome could readily penetrate such endothelial fenestrations to reach the target hepatocytes.

Alternatively or additionally, a liposome may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a liposome may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomes to hepatocytes.

In some embodiments, the size of a liposome is determined by the length of the largest diameter of the lipososme particle. In some embodiments, a suitable liposome has a size of or less than about 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 150 nm, 125 nm, 110 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50 nm. In some embodiments, a suitable liposome has a size no greater than about 250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm). In some embodiments, a suitable liposome has a size ranging from about 10-250 nm (e.g., ranging from about 10-225 nm, 10-200 nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, or 10-50 nm). In some embodiments, a suitable liposome has a size ranging from about 100-250 nm (e.g., ranging from about 100-225 nm, 100-200 nm, 100-175 nm, 100-150 nm). In some embodiments, a suitable liposome has a size ranging from about 10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, or 10-50 nm).

A variety of alternative methods known in the art are available for sizing of a population of liposomes. One such sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small ULV less than about 0.05 microns in diameter. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.

Pharmaceutical Compositions

To facilitate expression of mRNA in vivo, delivery vehicles such as liposomes can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

Provided liposomally-encapsulated or associated mRNAs, and compositions containing the same, may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein (e.g., enzyme) production.

Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, and/or intranasal administration.

Alternately or additionally, liposomally encapsulated mRNAs and compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection. Formulations containing provided compositions complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.

In some embodiments, provided liposomes and/or compositions are formulated such that they are suitable for extended-release of the mRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice day, daily or every other day. In a preferred embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually. Also contemplated are compositions and liposomes which are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release a mRNA over extended periods of time. Preferably, the extended-release means employed are combined with modifications made to the mRNA to enhance stability.

Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein and related methods for the use of such compositions as disclosed for example, in U.S. Provisional Application No. 61/494,882, filed Jun. 8, 2011, the teachings of which are incorporated herein by reference in their entirety. For example, lyophilized pharmaceutical compositions according to the invention may be reconstituted prior to administration or can be reconstituted in vivo. For example, a lyophilized pharmaceutical composition can be formulated in an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod or membrane) and administered such that the dosage form is rehydrated over time in vivo by the individual's bodily fluids.

Provided liposomes and compositions may be administered to any desired tissue. In some embodiments, the mRNA delivered by provided liposomes or compositions is expressed in the tissue in which the liposomes and/or compositions were administered. In some embodiments, the mRNA delivered is expressed in a tissue different from the tissue in which the liposomes and/or compositions were administered Exemplary tissues in which delivered mRNA may be delivered and/or expressed include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.

According to various embodiments, the timing of expression of delivered mRNAs can be tuned to suit a particular medical need. In some embodiments, the expression of the protein encoded by delivered mRNA is detectable 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or 72 hours in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and/or 7 days in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable after a month or longer after a single administration of provided liposomes or compositions.

The present invention can be used to deliver mRNA at various doses. In some embodiments, an mRNA is administered at a dose ranging from about 0.1-5.0 mg/kg body weight, for example about 0.1-4.5, 0.1-4.0, 0.1-3.5, 0.1-3.0, 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3, 0.3-5.0, 0.3-4.5, 0.3-4.0, 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, 0.3-0.5, 0.5-5.0, 0.5-4.5, 0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, or 0.5-1.0 mg/kg body weight. In some embodiments, an mRNA is administered at a dose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg body weight.

EXAMPLES

While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.

Example 1. Exemplary Liposome Formulations for mRNA Delivery and Expression

This example provides exemplary liposome formulations incorporating the cationic lipids described in this application, for example, cKK-E12, for effective delivery and expression of mRNA encoding therapeutic proteins in vivo.

Lipid Materials

In general, the formulations described herein are based on a multi-component lipid mixture of varying ratios employing one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and/or cholesterol-based lipids), and one or more PEGylated lipids designed to encapsulate various nucleic acid-based materials. As a non-limiting example, cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione) is used in various formulations described herein. Exemplary helper lipids include one or more of DSPC (1,2-di stearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyri stoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc. Exemplary PEGylated lipids include a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C₆-C₂₀ length, for example, PEG-2K. As non-limiting examples, liposome formulations used in various examples described herein include cKK-E12, DOPE, cholesterol and DMG-PEG2K at various ratios. For example, in some cases, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by weight. In other cases, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by weight. Unless otherwise specified, the below Examples include a mixture in the ratio of cKK-E 12:DOPE:cholesterol:DMG-PEG2K of approximately 40:30:25:5 by weight.

Messenger RNA Material

The formulations described herein may be used to deliver any mRNA, in particular, therapeutic mRNA. As used herein, a therapeutic mRNA refers to an mRNA that encodes a therapeutic protein. The formulations described herein can also be used to deliver any modified or unmodified mRNA, or mRNA with naturally occurring sequences or codon-optimized.

As non-limiting examples, human Factor IX (FIX), codon-optimized Firefly Luciferase (FFL), codon-optimized human argininosuccinate synthetase (ASS1) messenger RNA, codon-optimized human Survival of Motor Neuron 1(SMN) mRNA were synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5′ cap structure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA cap structures by viral and cellular proteins” J. Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail of, e.g., approximately 250 nucleotides in length (SEQ ID NO: 15) as determined by gel electrophoresis. Typically, 5′ and 3′ untranslated regions (UTR) are present in each mRNA product and are represented as X and Y, respectively. Example 5′ and 3′ UTR sequences are described below. The exemplary sequences of FIX, ASS1, and FFL mRNA used in the examples herein are listed below. Also shown are the 5′ and 3′ UTR sequences.

Human Factor IX (FIX) mRNA: (SEQ ID NO.: 1) XAUGCAGCGCGUGAACAUGAUCAUGGCAGAAUCACCAGGCCUCAUCACCA UCUGCCUUUUAGGAUAUCUACUCAGUGCUGAAUGUACAGUUUUUCUUGAU CAUGAAAACGCCAACAAAAUUCUGAGGCGGAGAAGGAGGUAUAAUUCAGG UAAAUUGGAAGAGUUUGUUCAAGGGAACCUUGAGAGAGAAUGUAUGGAAG AAAAGUGUAGUUUUGAAGAAGCACGAGAAGUUUUUGAAAACACUGAAAGA ACAACUGAAUUUUGGAAGCAGUAUGUUGAUGGAGAUCAGUGUGAGUCCAA UCCAUGUUUAAAUGGCGGCAGUUGCAAGGAUGACAUUAAUUCCUAUGAAU GUUGGUGUCCCUUUGGAUUUGAAGGAAAGAACUGUGAAUUAGAUGUAACA UGUAACAUUAAGAAUGGCAGAUGCGAGCAGUUUUGUAAAAAUAGUGCUGA UAACAAGGUGGUUUGCUCCUGUACUGAGGGAUAUCGACUUGCAGAAAACC AGAAGUCCUGUGAACCAGCAGUGCCAUUUCCAUGUGGAAGAGUUUCUGUU UCACAAACUUCUAAGCUCACCCGUGCUGAGGCUGUUUUUCCUGAUGUGGA CUAUGUAAAUUCUACUGAAGCUGAAACCAUUUUGGAUAACAUCACUCAAA GCACCCAAUCAUUUAAUGACUUCACUCGGGUUGUUGGUGGAGAAGAUGCC AAACCAGGUCAAUUCCCUUGGCAGGUUGUUUUGAAUGGUAAAGUUGAUGC AUUCUGUGGAGGCUCUAUCGUUAAUGAAAAAUGGAUUGUAACUGCUGCCC ACUGUGUUGAAACUGGUGUUAAAAUUACAGUUGUCGCAGGUGAACAUAAU AUUGAGGAGACAGAACAUACAGAGCAAAAGCGAAAUGUGAUUCGAAUUAU UCCUCACCACAACUACAAUGCAGCUAUUAAUAAGUACAACCAUGACAUUG CCCUUCUGGAACUGGACGAACCCUUAGUGCUAAACAGCUACGUUACACCU AUUUGCAUUGCUGACAAGGAAUACACGAACAUCUUCCUCAAAUUUGGAUC UGGCUAUGUAAGUGGCUGGGGAAGAGUCUUCCACAAAGGGAGAUCAGCUU UAGUUCUUCAGUACCUUAGAGUUCCACUUGUUGACCGAGCCACAUGUCUU CGAUCUACAAAGUUCACCAUCUAUAACAACAUGUUCUGUGCUGGCUUCCA UGAAGGAGGUAGAGAUUCAUGUCAAGGAGAUAGUGGGGGACCCCAUGUUA CUGAAGUGGAAGGGACCAGUUUCUUAACUGGAAUUAUUAGCUGGGGUGAA GAGUGUGCAAUGAAAGGCAAAUAUGGAAUAUAUACCAAGGUAUCCCGGUA UGUCAACUGGAUUAAGGAAAAAACAAAGCUCACUUAAY Codon-Optimized Human Argininosuccinate Synthetase (ASS1) mRNA: (SEQ ID NO.: 2) XAUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACA CCAGCUGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCC UACCUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAA GGCCCUGAAGCUGGGCGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCG AGUUCGUGGAGGAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUAC GAGGACCGCUACCUGCUGGGCACCAGCCUGGCCCGCCCCUGCAUCGCCCG CAAGCAGGUGGAGAUCGCCCAGCGCGAGGGCGCCAAGUACGUGAGCCACG GCGCCACCGGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUAC AGCCUGGCCCCCCAGAUCAAGGUGAUCGCCCCCUGGCGCAUGCCCGAGUU CUACAACCGCUUCAAGGGCCGCAACGACCUGAUGGAGUACGCCAAGCAGC ACGGCAUCCCCAUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAG AACCUGAUGCACAUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAA CCAGGCCCCCCCCGGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCC CCAACACCCCCGACAUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUG AAGGUGACCAACGUGAAGGACGGCACCACCCACCAGACCAGCCUGGAGCU GUUCAUGUACCUGAACGAGGUGGCCGGCAAGCACGGCGUGGGCCGCAUCG ACAUCGUGGAGAACCGCUUCAUCGGCAUGAAGAGCCGCGGCAUCUACGAG ACCCCCGCCGGCACCAUCCUGUACCACGCCCACCUGGACAUCGAGGCCUU CACCAUGGACCGCGAGGUGCGCAAGAUCAAGCAGGGCCUGGGCCUGAAGU UCGCCGAGCUGGUGUACACCGGCUUCUGGCACAGCCCCGAGUGCGAGUUC GUGCGCCACUGCAUCGCCAAGAGCCAGGAGCGCGUGGAGGGCAAGGUGCA GGUGAGCGUGCUGAAGGGCCAGGUGUACAUCCUGGGCCGCGAGAGCCCCC UGAGCCUGUACAACGAGGAGCUGGUGAGCAUGAACGUGCAGGGCGACUAC GAGCCCACCGACGCCACCGGCUUCAUCAACAUCAACAGCCUGCGCCUGAA GGAGUACCACCGCCUGCAGAGCAAGGUGACCGCCAAGUGAY Codon-Optimized Firefly Luciferase (FFL) mRNA: (SEQ ID NO.: 3) XAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCAC UCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUAC GCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGA CAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUA UGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAG AAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGU GGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACA GCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUG CAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAU CAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCU UCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCC GAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGG CAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUG UCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCC GACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUU CACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACC GCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAA UCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCU CAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGG CGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUA CCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCU GAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGC CCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGU GUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGG CUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCU GGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUC AUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGC CCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACG CCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCA GUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGA CUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUG UGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGC AAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGU GUAAY Codon-Optimized Human Survival of Motor Neuron 1(SMN) mRNA: (SEQ ID NO: 4) XAUGGCCAUGAGCAGCGGAGGCAGCGGCGGAGGAGUGCCCGAGCAGGAGG ACAGCGUGCUGUUCAGGAGAGGCACCGGCCAGAGCGAUGACAGCGAUAUC UGGGACGAUACCGCUCUGAUCAAGGCCUACGACAAGGCCGUGGCCAGCUU CAAGCACGCCCUGAAAAACGGCGACAUCUGCGAGACCAGCGGCAAGCCCA AGACAACCCCCAAGAGAAAGCCCGCCAAGAAGAAUAAGAGCCAGAAAAAG AACACCGCCGCCAGCCUGCAGCAGUGGAAGGUGGGCGACAAGUGCAGCGC CAUCUGGAGCGAGGACGGCUGCAUCUACCCCGCCACCAUCGCCAGCAUCG ACUUCAAGAGAGAGACCUGCGUGGUCGUGUACACCGGCUACGGCAACAGA GAGGAGCAGAACCUGAGCGACCUGCUGAGCCCCAUUUGUGAGGUGGCCAA UAACAUCGAACAGAACGCCCAGGAGAACGAGAAUGAAAGCCAGGUGAGCA CCGACGAGAGCGAGAACAGCAGAUCUCCUGGCAACAAGAGCGACAACAUC AAGCCUAAGUCUGCCCCUUGGAACAGCUUCCUGCCCCCUCCUCCACCCAU GCCCGGACCCAGACUGGGACCCGGAAAACCUGGCCUGAAGUUCAACGGAC CACCUCCCCCUCCACCUCCUCCCCCACCUCAUCUCCUGAGCUGCUGGCUG CCACCCUUCCCCAGCGGACCCCCUAUCAUCCCACCACCCCCUCCCAUCUG CCCCGACAGCCUGGACGACGCCGAUGCCCUGGGCAGCAUGCUGAUCAGCU GGUACAUGAGCGGCUACCACACAGGAUACUACAUGGGCUUCAGACAGAAC CAGAAGGAGGGCAGAUGCUCCCACUCCCUGAACUGAY 5′ and 3′ UTR Sequences X (5′ UTR Sequence) = (SEQ ID NO.: 5) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAG ACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGC GGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG Y (3′ UTR Sequence) = (SEQ ID NO.: 6) CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAG UUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC AAGCU or (SEQ ID NO.: 7) GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGU UGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCA AAGCU C-terminal His₁₀ Codon-Optimized Human CFTR mRNA (“His₁₀” disclosed as SEQ ID NO: 11): (SEQ ID NO.: 8) XAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCU UCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAG UUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUC GGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACC CGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUC UACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCU GUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAAC GGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUC AGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAU GCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAAC UCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUG CUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUU CGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGG AGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUG GCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCA GAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCG AAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAG AUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGC GUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCG UUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUC CGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGU GACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAG CGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUG GAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUU UUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACA ACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUC UCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAG GGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCC UCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAA CACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGG AACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAU ACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUC GCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGG AGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAG AUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAA AAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAG AAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCC UGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUG CAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUU CGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGC ACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAG AAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAG UAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAA CUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAG CGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCC CCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGC AAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUU CACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGC GAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCG GACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUC UUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUU GCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUC UCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUG CUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAA CAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUU ACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGA GGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCA UAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGC UCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUG GAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAU CGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUG UCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUC UUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCC UAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCG CCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAU CUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCA GAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUA UCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUG ACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUC GAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCA UCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAG AAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAA GGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGG CAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGC AUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAA AUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGA UCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGG AAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUU CCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGA AAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGA AAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCA CAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUC UUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUC AUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUG UGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCG AAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAG AGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUU UCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCU UGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUCACCAUCAC CAUCACCAUCACCAUCACCAUUAAY Codon-Optimized Human CFTR mRNA: (SEQ ID NO.: 9) XAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCU UCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAG UUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUC GGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACC CGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUC UACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCU GUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAAC GGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUC AGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAU GCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAAC UCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUG CUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUU CGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGG AGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUG GCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCA GAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCG AAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAG AUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGC GUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCG UUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUC CGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGU GACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAG CGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUG GAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUU UUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACA ACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUC UCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAG GGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCC UCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAA CACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGG AACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAU ACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUC GCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGG AGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAG AUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAA AAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAG AAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCC UGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUG CAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUU CGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGC ACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAG AAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAG UAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAA CUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAG CGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCC CCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGC AAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUU CACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGC GAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCG GACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUC UUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUU GCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUC UCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUG CUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAA CAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUU ACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGA GGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCA UAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGC UCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUG GAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAU CGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUG UCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUC UUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCC UAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCG CCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAU CUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCA GAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUA UCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUG ACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUC GAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCA UCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAG AAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAA GGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGG CAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGC AUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAA AUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGA UCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGG AAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUU CCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGA AAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGA AAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCA CAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUC UUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUC AUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUG UGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCG AAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAG AGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUU UCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCU UGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAAY Codon Optimized Human CFTR mRNA coding sequence with a Growth Hormone Leader Sequence (italisized and underlined) (SEQ ID NO: 10) AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUG CCUGCCCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC A UGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUC UCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUU GUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGG AGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCG AAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUA CGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGU UGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGG AGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAG AACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGC AGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUC UCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCU UAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCG UGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAG CUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGC AUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGA GAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAA AACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAU GAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGU AUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUU GUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCG CAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGA CACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCG AUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGA GUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUU GGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAAC AACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUC CCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGG GACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUC UUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACA CUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAA CCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUAC AGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGC CGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAG GGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAU UUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAA AGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAA UCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUG AUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCA AAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCG ACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCAC CGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAA GCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUA UCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACU CCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCG CAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCC GGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAA UCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCA CCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGA AUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGA CUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUU UGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGC GUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUC GUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCU UGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACA AUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUAC AUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGG ACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUA AGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUC AAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGA UGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCG UGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUC GCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUU GCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUA UCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCC UUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCU CCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGA UGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUC UCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGAC ACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGA UUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUC GACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAA UGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGG AUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCA AAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAU UAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAU CGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUC CAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAA AGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCC GAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAA GUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAA ACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACA AGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUU CUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAU CAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUG AGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAA GAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAG AUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUC CACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUG AAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA

Aliquots of 50 mg/mL ethanolic solutions of cKK-E12, DOPE, Chol and DMG-PEG2K were mixed in a molar ratio of 40:30:25:5 and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of FIX, ASS1, or FFL mRNA was prepared from a 1 mg/mL stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. The final concentration of FIX mRNA was approximately 0.77 mg/mL FIX mRNA (encapsulated), Z_(ave)=76 nm, PDI=0.08. The final concentration of ASS1 mRNA was approximately 0.64 mg/mL ASS1 mRNA (encapsulated), Z_(ave)=78 nm (Dv(50)=46 nm; Dv(90)=96 nm). The final concentration ofFFL mRNA was approximately 1.31 mg/mL FFL mRNA (encapsulated), Z_(ave)=75 nm, PDI—0.11. The final concentration of SMN mRNA was approximately 1.85 mg/mL SMN mRNA (encapsulated). Average particle size (Z_(ave))=71 nm, (particle size for 50% of particles was 44 nm or less (Dv(50))=44 nm; and the particle size for 90% of the particles was 93n or less (Dv(90)=93 nm)).

Example 2. Administration of mRNA-Loaded Liposome Nanoparticles

This example illustrates exemplary methods of administering mRNA-loaded liposome nanoparticles and methods for analyzing delivered mRNA and subsequently expressed protein in various target tissues in vivo.

All studies were performed using male CD-1 mice of approximately 6-8 weeks of age at the beginning of each experiment. Samples were introduced by a single bolus tail-vein injection of an equivalent total dose of 1.0 mg/kg (or otherwise specified) of encapsulated FIX, FFL or ASS1 mRNA. Mice were sacrificed and perfused with saline at the designated time points.

Various organ tissues such as the liver, spleen, kidney and heart of each mouse was harvested, apportioned into separate parts, and stored in either 10% neutral buffered formalin or snap-frozen and stored at −80° C. for analysis.

All animals were euthanized by CO₂ asphyxiation at designated time points post dose administration (±5%) followed by thoracotomy and terminal cardiac blood collection. Whole blood (maximal obtainable volume) was collected via cardiac puncture on euthanized animals into serum separator tubes, allowed to clot at room temperature for at least 30 minutes, centrifuged at 22° C.±5° C. at 9300 g for 10 minutes, and the serum extracted. For interim blood collections, approximately 40-50 μL of whole blood was collected via facial vein puncture or tail snip. Samples collected from non-treatment animals were used as a baseline ASS1 levels for comparison to study animals.

Enzyme-Linked Immunosorbent Assay (ELISA) Analysis

A. Human FIX ELISA

Quantification of FIX protein was performed following procedures reported for human FIX ELISA kit (AssayMax, Assay Pro, Catalog # EF1009-1).

B. Human ASS1 ELISA

Standard ELISA procedures were followed employing mouse anti-ASS1 2D1-2E12 IgG as the capture antibody with rabbit anti-ASS1 #3285 IgG as the secondary (detection) antibody (Shire Human Genetic Therapies). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was used for activation of the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The reaction was quenched using 2N H2SO4 after 20 minutes. Detection was monitored via absorption (450 nm) on a Molecular Device SpectraMax instrument. Untreated mouse serum and organs and human ASS1 protein were used as negative and positive controls, respectively.

IVIS Bioluminometer Measurements

To visual luminescence in treated mice, several steps were followed. Anesthesia using isoflurane vaporizer at 1-3% (usually @2.5%) was initially employed. Using a microsprayer, 50 μL/animal of luciferin in PBS was administered at 60 mg/mL via intratracheal/intranasal. Luciferin was allowed to distribute for 5-10 minutes. Animals were placed in an isoflurane chamber until anesthetized. Anesthetized animals were placed into the IVIS imaging chamber at dorsal recumbency and positioned into the manifold. Pictures of mice were taken. In these Examples, the acquisition settings providing highest sensitivity were: camera height at D level, F/Stop at f1, binning at high resolution, and exposure time at 5 minutes. Exposures were repeated up to 3 times (5, 10 and 15 minutes post Luciferin Injection).

In Situ Hybridization (ISH) Analysis

In situ hybridization was performed using “ZZ” probe technology. Probes were generated based on codon-optimized sequence of human messenger RNA. Tissues were fixed for 24-48 hours in 10% neutral buffered formalin and embedded in paraffin. Positive detection of desired mRNA was achieved through 6 consecutive amplification steps followed by chromagenic visualization using 3,3′-diaminobenzidine (DAB). Positive signal was compared to that of untreated mouse.

Example 3. Highly Effective In Vivo Production of Therapeutic Proteins

This example demonstrates highly efficient and sustained production of proteins encoded by mRNA delivered by liposomes incorporating the cationic lipids described herein (e.g., cKK-E12) in serum and various organ tissues.

In Vivo Human FIX Protein Production Results

The production of human FIX protein via hFIX mRNA-loaded cKK-E12-based lipid nanoparticles was tested in CD-1 mice as a single, bolus intravenous injection. FIG. 1 represents the amount of human FIX protein detected via ELISA when treating mice with human FIX mRNA-loaded cKK-E12-based lipid nanoparticles as compared to a C12-200-based lipid nanoparticle encapsulating hFIX mRNA. The mice were sacrificed twenty-four hours post-injection and organs were harvested (as described above).

C12-200-based lipid nanoparticles have been shown to be an effective vehicle to deliver and express mRNA in vivo (see, PCT Application Publication NO. WO2012170930, the disclosure of which is hereby incorporated by reference). Surprisingly, as represented in FIG. 1, cKK-E12 based lipid nanoparticles are even more effective in delivering human FIX mRNA in vivo, resulting in close to 50% higher protein expression detected in the plasma of the treated mice, as compared to C12-200-based lipid nanoparticles.

FIG. 2 shows the results of a dose-response experiment as represented by the amount of human FIX protein detected via ELISA when treating mice with human FIX mRNA-loaded cKK-E12-based lipid nanoparticles at various doses. The mice were bled at 6 hours and sacrificed twenty-four hours post-injection and organs were harvested (as described above).

A clear dose response was achieved when measuring liver levels of human FIX protein. The dosing range was from 0.10-3.0 mg/kg of encapsulated human FIX mRNA. These data demonstrate the ability of the lipid nanoparticles to efficiently deliver messenger RNA, release the payload and process this exogenous mRNA via translation to produce human FIX protein, which is then subsequently secreted into the bloodstream. Levels of human FIX protein are well above therapeutic levels (>100 ng/mL plasma) and surpass normal physiological levels (˜5 ug/mL plasma) when dosing at 1.0 mg/kg or greater. Further, the plasma residence time of this human protein is sustained through at least 24 hours post administration.

In Vivo Human ASS1 Protein Production Results

The production of human ASS1 protein via codon-optimized hASS1 mRNA-loaded cKK-E12-based lipid nanoparticles was tested in CD-1 mice as a single, bolus intravenous injection. FIG. 3 represents the amount of human ASS1 protein detected via ELISA when treating mice with human ASS1 mRNA-loaded cKK-E12-based lipid nanoparticles at various doses. The mice were sacrificed twenty-four hours post-injection and organs were harvested (as described above).

A clear dose response was achieved when measuring liver levels of human ASS1 protein. As shown in Table 5, the dosing range was from 0.10-2.0 mg/kg of encapsulated human ASS1 mRNA in cKK-E12 lipid nanoparticles. These data demonstrate the ability of the lipid nanoparticles to accumulate in the liver and release the mRNA payload and the liver to process this exogenous mRNA via translation to produce human ASS1 protein.

TABLE 5 Raw values of human ASS1 protein as measured via ELISA analysis (as depicted in FIG. 1). Codon-optimized human ASS1 mRNA was delivered via cKK-E12-based lipid nanoparticles. Doses are based on encapsulated ASS1 mRNA. Values are depicted as nanogram of human ASS1 protein per milligram total protein in liver. Dose Encapsulated Human ASS1 mRNA (mg/kg) ASS1 Protein (ng/mg total protein) 0.10 BLD 0.30 BLD 0.60 546 1.0 1388 2.0 3371 BLD = Below Limit of Detection for ELISA.

While the sensitivity of the ELISA has limitations at lower values, western blot analysis allows for clear visualization of the human ASS1 protein at lower doses (0.3-3.0 mg/kg) (see FIG. 4). FIG. 4 depicts a comparison of human ASS1 protein levels in liver as a function of dose via western blot analysis upon a single intravenous dose of human ASS1 mRNA-encapsulated cKK-E12 lipid nanoparticles. CD1 mice were sacrificed at 24 hours post-administration and livers were harvested and analyzed as described above.

To further understand the ability of ASS1 mRNA-encapsulated lipid nanoparticles to facilitate the delivery of mRNA to selected organs (liver), a pharmacokinetic analysis was performed, monitoring human ASS1 protein levels in the liver over a one week time period. FIG. 5 depicts the quantity of human ASS1 protein detected in the liver at various time points up to 7 days after administration of human ASS1-loaded lipid nanoparticles (cKK-E12). This was accomplished as a single dose (1.0 mg/kg encapsulated mRNA) given intravenously.

In this case, we observed a maximum serum level of human ASS1 protein at approximately 24-48 hours post-administration. Measurable levels of protein were still observed 1 week post-administration as determined by both ELISA and western blot (FIGS. 5 and 6, respectively). FIG. 6 depicts a comparison of human ASS1 protein levels in liver over time via western blot analysis upon a single intravenous dose of human ASS1 mRNA-encapsulated lipid nanoparticles (1.0 mg/kg dose).

Direct detection of the active pharmaceutical ingredient (ASS1 mRNA) in the livers of the treated mice was achieved using in situ hybridization (ISH) based methods. As demonstrated in FIGS. 7 & 8, the exogenous human ASS1 messenger RNA could be detected in high levels at the earliest time point tested (30 minutes) and the signal remained strong for 48 hours after dosing. Further, human ASS1 mRNA was still detectable 72 hours post-administration.

In addition to ISH, detection of the resulting human ASS1 protein was achieved using immunohistochemical (IHC) means. Using a mouse monoclonal antibody (02D2-2E12) for specific binding, the presence of target human ASS1 protein in the cytoplasm of hepatocytes of treated livers can be readily observed. FIG. 9 shows the immunohistochemical staining of human ASS1 protein in treated mouse livers 24 hours after administration.

In Vivo Delivery of FFL mRNA Via Nebulization

To assess whether additional routes of delivery were feasible, FFL mRNA was encapsulated in cKK-E12 liposomes and those liposomes were nebulized. As shown in FIG. 10, it is possible to efficiently nebulize cKK-E12 based lipid nanoparticles encapsulating mRNA. FIG. 10 represents mice treated with luciferin 24 hours after exposure to nebulized FFL mRNA loaded cKK-E12 lipid nanoparticles.

Example 4. CNS Delivery of hSMN-1 mRNA

This example provides an exemplary cKK-E12 liposome formulations for effective delivery and expression of mRNA in the CNS. Specifically, the example demonstrates that delivery of human survival of motor neuron-1 (hSMN-1) mRNA into various tissues of the brain and spinal cord.

Messenger RNA Material

Codon-optimized human Survival of Motor Neuron-1(hSMN-1) messenger RNA (see SEQ ID NO: 4) was synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5′ cap structure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA cap structures by viral and cellular proteins” J. Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail of approximately 250 nucleotides in length (SEQ ID NO: 15) as determined by gel electrophoresis. The 5′ and 3′ untranslated regions present in each mRNA product are represented as X and Y, respectively and defined as stated in Example 1.

Formulation Protocol

Lipid nanoparticles (LNP) were formed via standard ethanol injection methods (Ponsa, M.; Foradada, M.; Estelrich, J. “Liposomes obtained by the ethanol injection method” Int. J. Pharm. 1993, 95, 51-56). For the various lipid components, a 50 mg/ml ethanolic stock solutions was prepared and stored at −20° C. In preparation of the cKK-E12 lipid nanoparticle formulation listed in Table 6, each indicated lipid component was added to an ethanol solution to achieve a predetermined final concentration and molar ratio, and scaled to a 3 ml final volume of ethanol. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of hSMN-1 mRNA was prepared from a 1 mg/ml stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered and dialysed against 1×PBS (pH 7.4), concentrated and stored between 2-8° C. SMN-1 mRNA concentration was determined via the Ribogreen assay (Invitrogen). Encapsulation of mRNA was calculated by performing the Ribogreen assay with and without the presence of 0.1% Triton-X 100. Particle sizes (dynamic light scattering (DLS)) and zeta potentials were determined using a Malvern Zetasizer instrument in 1×PBS and 1 mM KCl solutions, respectively.

TABLE 6 Exemplary cKK-E12 Lipid Nanoparticle formulation Final Molar mRNA Formu- Ratio Concen- Zeta lations Components of lipids tration Parameters 1 cKK-E12 40:30:25:5 1.8 mg/ml Z_(ave) = 72 nm; DOPE Dv₍₅₀₎ = 49 nm; Cholesterol Dv₍₉₀₎ = 90 nm DMG-PEG-2K hSMN-1 mRNA

Intrathecal Administration of mRNA Loaded Liposome Nanoparticles

All in vivo studies were performed using either rats or mice of approximately 6-8 weeks of age at the beginning of each experiment. At the start of the experiment, each animal was anesthetized with isoflurane (1-3%, to effect) by inhalation. Once anesthetized, each animal was shaved at the exact injection site (L4-L5 or L5-L6). Following insertion of the needle, reflexive flick of the tail was used to indicate puncture of the dura and confirm intrathecal placement. Each animal received a single bolus intrathecal injection of the test formulation listed in Table 6. All animals were sacrificed 24 hours post injection and perfused with saline.

Isolation of Organ Tissues for Analysis

All animals had the whole brain and spinal cord harvested. The brain was cut longitudinally and placed in one histology cassette per animal. The whole spinal cord was stored ambient in a 15 ml tube containing 10% neutral buffered formalin (NBF) for at least 24 hours and no more than 72 hours before transfer into 70% histology grade alcohol solution. Each spinal cord sample was cut into cervical, thoracic and lumbar sections. Each spinal cord section cut in half and both halves were placed in individual cassettes per section (cervical, thoracic and lumbar) for processing. All three cassettes were embedded into one paraffin block per animal. When applicable, portions of brain and spinal cord were snap frozen and stored at −80° C.

hSMN-1 Western Blot Analysis

Standard western blot procedures were followed employing various antibodies that recognizes hSMN protein, such as: (A) anti-SMN 4F11 antibody at 1:1,000 dilution; (B) Pierce PA5-27309 a-SMN antibody at 1:1,000 dilution; and (C) LSBio C138149 a-SMN antibody at 1:1,000 dilution. For each experiment one microgram of hSMN mRNA was transfected into ˜1×10⁶ BHK-21 cells using Lipofectamine 2000. Cells were treated with OptiMem and harvested 16-18 hours post-transfection. Cell lysates were harvested, processed and loaded on to an 8-16% Tris Glycine gel. The gel was transferred using a PVDF membrane and treated with the respective primary antibody. Goat anti-mouse HRP antibody was used as the secondary antibody at 1:10,000 dilution for 45 minutes at room temperature followed by washing and development. The data demonstrates that each antibody tested showed a strong signal for hSMN-1 and was specific for human SMN, as indicated by an absence in a cross-reactive signal for untreated BHK cells (FIG. 11).

In Situ Hybridzation (ISH) Analysis

Tissue from each representative sample, was assayed for hSMN-1 mRNA using a manual in situ hybridization analysis, performed using RNAscope® (Advanced Cell Diagnostic) “ZZ” probe technology. Probes were generated based on the codon-optimized sequence of human SMN messenger RNA (SEQ ID NO: 4). Briefly, the RNAscope® assay is an in situ hybridication assay designed to visualize single RNA molecules per cell in formalin-fixed, paraffin-embedded (FFPE) tissue mounted on slides. Each embedded tissue sample was pretreated according to the manufacturers protocol and incubated with a target specific hSMN-1 RNA probe. The hSMN-1 probe was shown to be specific for human SMN-1 and had little to no cross reactivity with mouse or rat SMN-1. Once bound, the hSMN-1 probe is hybridized to a cascade of signal amplification molecules, through a series of 6 consecutive rounds of amplification. The sample was then treated with an HRP-labeled probe specific to the signal amplification cassette and assayed by chromatic visualization using 3,3′-diaminobenzidine (DAB). A probe specific for Ubiquitin C was used as the positive control. Positive SMN signal was compared to that of untreated and vehicle control treated rat or mouse tissue. Stained samples were visualized under a standard bright field microscope.

Immunohistochemical Analysis

Human SMN-1 mRNA-loaded lipid nanoparticles were administered to rats via intrathecal injection, and tissue samples collected and processed 24 hours post administration in accordance with the methods described above. Rat spinal tissue samples were then assayed for hSMN-1 protein expression. Briefly, fixed tissue embedded in paraffin was processed and placed on slides. The slides were dewaxed, rehydrated and antigen retrieval was performed using a pressure cooker with citrate buffer. Several blocking buffers were employed followed by primary antibody incubation overnight at 4° C., using the 4F11 antibody at a 1:2500 dilution. The resulting slides were washed and incubated at ambient temperature with the secondary antibody polymer followed by washing and subsequent chromagen development. The data demonstrates that in as little as 24 hours post intrathecal adminiatration of hSMN-1 mRNA, staining is observed for human SMN-1 protein when compared to no-treatment control (FIG. 13). This supports the previous findings which demonstrate delivery of hSMN-1 mRNA to the spinal tissue. Furthermore, the data demonstrates that once delivered to the cell hSMN-1 mRNA is effectively expressed to generate hSMN-1 protein.

Results

The data presented in this example demonstrates that intrathecal administration of hSMN-1 mRNA loaded liposomes (e.g., lipid or polymer-based nanoparticles) results in successful intracellular delivery of mRNA in neurons in the brain and spinal cord, including those difficult to treat cells, such as anterior horn cells and dorsal root ganglia.

The results have shown that mRNA encapsulated within a lipid nanoparticle (e.g., lipid nanoparticle comprising cKK-E12) can be effectively delivered to various tissues of the CNS following intrathecal administrations. Using the exemplary formulation disclosed in Table 6, mRNA was effectively delivered and internalized within various neurons of the spinal cord (FIGS. 12A-12C), as verified by in situ hybridization assay. Surprisingly, intracellular mRNA delivery was demonstrated in the difficult to reach neuronal cells of the anterior horn, located deep within the tissues of the spinal column (FIGS. 12A-12C). Little to no background was observed with mouse or rat SMN-1, indicating specificity for the human SMN-1 probe. Positive SMN signal was compared to that of untreated and vehicle control treated rat or mouse tissue. Stained samples were visualized under a standard bright field microscope.

These data demonstrates that the lipid or polymer nanoparticle based mRNA delivery approach described herein was able to successfully permeate the complex and dense cell membrane of the spinal cord neurons and deliver the mRNA payload for the production of encoded proteins inside neurons. It was particularly surprising that the mRNA delivery approach described herein was equally successful in permeating difficult to treat neurons such as anterior horn cell and dorsal root ganglia. Thus, the data presented herein demonstrates that lipid or polymer nanoparticles, such as those comprising cKK-E12, may serve as a promising option for delivering mRNA to neuronal cells in the treatment of a CNS disease. In particular, the present example demonstrates that hSMN mRNA loaded nanoparticles can be effectively delivered to neurons, including those difficult to treat motor neurons in the spinal cord, and can be used for the production of SMN protein and treatment of spinal muscular atrophy.

Example 5. In Vivo CO-CFTR—C-His₁₁ mRNA Delivery to CFTR Knockout Mice

Messenger RNA Synthesis. For the experiment, C-terminal His₁₀ tagged codon-optimized human cystic fibrosis transmembrane conductance regulator (CO-CFTR—C-His₁₀) (SEQ ID NO:8) (“His₁₀” disclosed as SEQ ID NO: 11) and non-tagged codon-optimized human CFTR (CO-CFTR) (SEQ ID NO:9) mRNA were synthesized by in vitro transcription from a plasmid DNA template using standard method. mRNAs used in this example and Example 6 were produced by IVT in which 25% of U residues were 2-thio-uridine and 25% of C residues were 5-methylcytidine.

Analysis of Human CFTR Protein Produced Via Intratracheal Administered mRNA-Loaded Nanoparticles.

For the study, CFTR knockout mice were used. CFTR mRNA formulation or vehicle control was introduced using a PARI Boy jet nebulizer. Mice were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.

PEI Formulation.

PEI formulation has been used to deliver CFTR mRNA to the lung and was used as a control in this experiment. Polymeric nanoparticle formulations with 25 kDa branched PEI were prepared as follows. The required amount of mRNA was diluted just before application in water for injection (Braun, Melsungen) to a total volume of 4 ml and added quickly to 4 ml of an aqueous solution of branched PEI 25 kDa using a pipette at an N/P ratio of 10. The solution was mixed by pipetting up and down ten times and nebulized as two separate 4.0 ml fractions one after another to the mouse lungs using the indicated nebulizer.

cKK-E12 Formulation.

For the lipid-based nanoparticle experiment, a lipid formulation was created using CO-CFTR—C-His₁₀ RNA in a formulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg)). The solution was nebulized to the mouse lungs using the indicated nebulizer.

Nebulization (Aerosol) Administration of Human CO-CFTR—C-His₁₀ mRNA.

CFTR test materials were administered by a single aerosol inhalation via PARI Boy jet nebulizer (nominal dose volume of up to 8 mL/group). The test material was delivered to a box containing the whole group of animals (n=4) and connected to oxygen flow and scavenger system.

Administration of Human CO-CFTR-C-His₁₀ mRNA.

CFTR mRNA was prepared in the manner described above. Four CFTR knockout mice were placed in an aerosol chamber box and exposed to 2 mg total codon optimized unmodified human CFTR mRNA (comprising the coding sequence of SEQ ID NO: 8) via nebulization (Pari Boy jet nebulizer) over the course of approximately one hour. Mice were sacrificed 24 hours post-exposure.

Euthanasia.

Animals were euthanized by CO₂ asphyxiation at representative times post-dose administration (+5%) followed by thoracotomy and exsanguinations. Whole blood (maximal obtainable volume) was collected via cardiac puncture and discarded.

Perfusion.

Following exsanguination, all animals underwent cardiac perfusion with saline. In brief, whole body intracardiac perfusion was performed by inserting 23/21 gauge needle attached to 10 mL syringe containing saline set into the lumen of the left ventricle for perfusion. The right atrium was incised to provide a drainage outlet for perfusate. Gentle and steady pressure was applied to the plunger to perfuse the animal after the needle had been positioned in the heart. Adequate flow of the flushing solution was ensured when the exiting perfusate flows clear (free of visible blood) indicating that the flushing solution has saturated the body and the procedure was complete.

Tissue Collection.

Following perfusion, all animals had their lungs (right and left) harvested. Both (right and left) lungs were snap frozen in liquid nitrogen and stored separately at nominally −70° C.

Expression of Human CFTR in CO-CFTR-C-His₁₀ in CFTR Knockout Mice.

CFTR expression was detected by Western blot analysis of tissue lysate collected from CFTR mRNA-treated mouse lungs. Mature “C” band was detected in left and right lungs of all treated mice, for both the cKK-E12-based and PEI-based formulations (FIG. 14). Expression of the mature “C” band was verified by comparison with lysate collected from HEK 293T human CO-CFTR—C-His₁₀ positive cells. In contrast, no detectable signal was observed in lysate collected from wild type untreated control mice (FIG. 14). Taken together, these data suggest that cKK-E12 may be used to deliver mRNA (e.g., CFTR mRNA) to the lung via, e.g., inhalation, as effectively as or even better than PEI based formulations.

Example 6: In Vivo Expression in the Lung

This example further desmonstrates successful in vivo expression in the lung following aerosol delivery of mRNA-loaded ckk-E12 based nanoparticles. All studies were performed using pigs of the German Landrace, obtained from Technical University Munich, Weihenstephan, Germany. The pigs had a body weight ranging from 35-90 kg. FFL/CO-CFTR—C-His10 mRNA formulation or vehicle control was introduced using a Pari jet nebulizer. Pigs were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.

Messenger RNA Synthesis.

In the example, codon optimized fire fly luciferase (CO-FFL) mRNA was synthesized by in vitro transcription from plasmid DNA templates.

cKK-E12 Formulation.

For the lipid-based nanoparticle experiment, a lipid formulation was created using 1 mg FFL+9 mg of CO-CFTR—C-His₁₀ mRNA encapsulated in a formulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 40:30:25:5 (mol ratio). The solution was nebulized to the Pig lungs using the indicated nebulizer.

Aerosol Application.

The aerosol (Saline or CO-FFL cKK-E12 formulation) was nebulized and inhaled into the anaesthetized pig. Sedation in pigs was initiated by premedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg body weight, atropine 0.1 mg/kg body weight and followed by insertion of an intravenous line to the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg/kg body weight as required. Anesthesia was maintained by isoflurane (2-3%) with 1% propofol bolus injection at 4 to 8 mg/kg body weight to enhance anesthesia as required. Duration of the anesthesia was approximately 1-3 hrs. Pigs were killed with bolus injection of pentobarbital (100 mg/kg body weight) and potassium chloride via the lateral ear vein. Lungs were excised and tissue specimens were collected from various lung regions followed by incubation in cell culture medium overnight. The stored samples were subjected to bioluminescence detection.

Bioluminescence Analysis.

For measurement of luciferase activity, tissue specimens were either homogenized and analyzed in a tube luminometer or incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI. The data illustrate that a strong bioluminescence signal was observed for each of the (A)CO-FFL/CO-CFTR—C-His₁₀ mRNA treated pigs, when compared to (B) control lung tissue samples from control pigs (Saline vehicle control) (FIGS. 15 A&B).

These data illustrate that FFL/CFTR mRNA were successfully delivered to and expressed in the lung by aerosol administration of a cKK-E12 based lipid formulation.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: 

We claim:
 1. A method of delivery of messenger RNA (mRNA) in vivo, comprising administering to a subject in need of delivery a composition comprising an mRNA encoding a protein that is argininosuccinate synthetase (ASS1), encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in vivo; wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein: p is an integer of between 1 and 9, inclusive; each instance of R² is independently hydrogen or optionally substituted C₁₋₆ alkyl; each instance of R⁶ and R⁷ is independently a group of the formula (i), (ii), or (iii); Formulae (i), (ii), and (iii) are:

wherein: each instance of R′ is independently hydrogen or optionally substituted alkyl; X is O, S, or NR^(X), wherein R^(X) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; Y is O, S, or NR^(Y), wherein R is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; R^(P) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and R^(L) is optionally substituted C1-50 alkyl, optionally substituted C₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionally substituted heteroC₁₋₅₀ alkyl, optionally substituted heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.
 2. The method of claim 1, wherein the cationic lipid is cKK-E12:


3. The method of claim 1, wherein the liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.
 4. The method of claim 3, wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG-dieleeyl-(1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)).
 5. The method of claim 3, wherein the one or more cholesterol-based lipids are cholesterol and/or PEGylated cholesterol.
 6. The method of a claim 3, wherein the liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.
 7. The method of claim 1, wherein the liposome has a size less than about 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm.
 8. The method of claim 1, wherein the composition is administered intravenously.
 9. The method of claim 1, wherein the expression of the argininosuccinate synthetase (ASS1) protein encoded by the mRNA is detectable in liver.
 10. The method of claim 1, wherein the mRNA comprises one or more modified nucleotides that are pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and/or 2-thiocytidine.
 11. The method of claim 1, wherein the mRNA is unmodified.
 12. A method of treating citrullinemia, type I comprising administering to subject in need of treatment a composition comprising an mRNA encoding a therapeutic protein that is argininosuccinate synthetase (ASS1) encapsulated within a liposome such that the administering of the composition results in the expression of the ASS1 protein encoded by the mRNA in one or more tissues affected by the citrullinemia, type I; wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein: p is an integer of between 1 and 9, inclusive; each instance of R² is independently hydrogen or optionally substituted C₁₋₆ alkyl; each instance of R⁶ and R⁷ is independently a group of the formula (i), (ii), or (iii); Formulae (i), (ii), and (iii) are:

wherein: each instance of R′ is independently hydrogen or optionally substituted alkyl; X is O, S, or NR^(X), wherein R^(X) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; Y is O, S, or NR^(Y), wherein R^(Y) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; R^(P) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and R^(L) is optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionally substituted heteroC₁₋₅₀ alkyl, optionally substituted heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.
 13. The method of claim 12, wherein the cationic lipid is cKK-E12:


14. A composition for delivery of messenger RNA (mRNA) comprising an mRNA encoding a protein that is argininosuccinate synthetase (ASS1) encapsulated within a liposome, wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein: p is an integer of between 1 and 9, inclusive; each instance of R² is independently hydrogen or optionally substituted C₁₋₆ alkyl; each instance of R⁶ and R⁷ is independently a group of the formula (i), (ii), or (iii); Formulae (i), (ii), and (iii) are:

wherein: each instance of R′ is independently hydrogen or optionally substituted alkyl; X is O, S, or NR^(X), wherein R^(X) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; Y is O, S, or NR^(Y), wherein R is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; R^(P) is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally substituted C2-50 alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionally substituted heteroC₁₋₅₀ alkyl, optionally substituted heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.
 15. The composition of claim 14, wherein the cationic lipid is cKK-E12:


16. The composition of claim 14, wherein the liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.
 17. The composition of claim 16, wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)).
 18. The composition of claim 16, wherein the liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.
 19. The composition of claim 16, wherein the cationic lipid constitutes about 30-50% of the liposome by molar ratio.
 20. The composition of claim 14, wherein the liposome has a size less than about 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm.
 21. The composition of claim 14, wherein the composition is formulated for intravenous administration.
 22. The composition of claim 16, wherein the one or more cholesterol-based lipids are cholesterol and/or PEGylated cholesterol.
 23. The composition of claim 14, wherein the mRNA comprises one or more modified nucleotides comprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and/or 2-thiocytidine.
 24. The composition of claim 14, wherein the mRNA is unmodified. 